Non-tuberculous mycobacteria (NTM) are also referred to as mycobacteria other than tuberculosis (MOTT). These bacteria are ubiquitous in the environment and their distribution varies depending on environmental microbial distribution, climate and weather condition. Transmission occurs by aerosols for example during bathing and steaming. Over time the numbers of species and burden have been increasing. This includes saprophytes and emergence of species that cause diseases to human and animals. In general Mycobacterium avium complex (MAC) has been the most common isolated group of NTM. These bacteria have commonly been isolated from among patients presumed to have pulmonary Tuberculosis Non-tuberculous mycobacteria cause a variety of clinical conditions from localized infection to disseminated disease. Commonly they have been isolated from patients with atypical pulmonary TB. This condition is also known as NTM Pulmonary Disease (NTMPD) and it is clinically similar to TB. NTM-PD cases have remained undiagnosed or misdiagnosed for TB. This poses a high risk of unsuccessful treatment outcomes and long hospitalization. Management of NTM requires Healthcare Workers (HCW) with expertise in the field because despite of long courses of treatment required, treatment regimen also varies depending on the infecting species. Treatment of Mycobacteria avium Complex (MAC) and Mycobacteria abscessus Complex (MABC) mostly relies on the use of macrolides and aminoglycosides. Problem of NTM-PD is not yet well addressed in Tanzania. Success in detection and management of cases is largely limited by the level of awareness among HCWs and drug susceptibility of circulating species. The current study assessed knowledge gap, identify circulating NTM species as a single or co-infections with TB and their drug susceptibility. This study adopted a cross-sectional design. NTM-PD awareness was carried out on HCWs from Health facilities (HFs) in the four administrative Regions that make the Northern zone of Tanzania; Tanga, Arusha, Manyara and Kilimanjaro. Four cadres of HCWs from TB clinics were conveniently interviewed using standardized questionnaire. These included; clinicians, nurses, laboratory personnel and pharmacists. A list of HCWs for each cadre in each HF provided the sampling frame for the study. In a situation where more than one HCWs per cadre met inclusion criteria for survey in a particular HF, simple random selection was applied. Analysis of culture positive isolates was carried out at the Central TB Reference Laboratory. These included sample selection, storage, DNA extraction, speciation, drug susceptibility testing (DST). Isolates that were positive for para-nitrobenzoic acid (PNB) and all isolates from Kibong’oto zonal laboratory received between November, 2019 and August, 2020 were screened for NTMs. GenoLyse® protocol was used to extract DNA that were stored at – 200C till analysis. DNA amplification was done on the GTQ Cycler 96. Mycobacterium species were identified using GenoType® Mycobacterium CM/AS and GenoType® NTM-DR protocol. GenoType® NTM-DR was used to determine mutations that lead to drug resistance. An average score for awareness on Pulmonary NTM was 24.1% (n=120; 95% CI: 10.5-37.7 STD: = 11.72), for which the highest was 61% and the lowest was 3%. Only 5 (4%) of all participants had a fair level of awareness (scored 50 to 74%) while all the remaining had poor level of awareness. A strong relationship was observed between history of attending training in which NTM was a topic of study and high level of awareness (p<0.05). There was no statistical significant difference among the two genders. Mean awareness score of clinicians, laboratory staff, pharmacy staff and nurses were at 26% (STD: 13), 26% (STD: 11), 22% (STD: 14) and 21% (STD: 10), respectively. Level of education did not have statistically significant impact on the level of awareness in this study. Of the four regions; Kilimanjaro had the highest average awareness score of 26% (STD: 12.9) followed by Tanga 25% (STD: 11.7) and Manyara 24% (STD: 11.7) while Arusha had the lowest mean awareness score of 18% (STD: 7.5). A total of 188 mycobacteria isolates were GenoType® d for NTM species. Of these, 179 were positive for mycobacteria and the remaining were negative. Among the positive, 24 (13.4%) were NTM while others were MTBC. Fifteen (62.5%) of the NTM could be GenoType® d to species level. Predominant NTM groups were six (25%) MAC and four (16.7%) were MABC. Four (16.7%) isolates were M. intracellulare, 3 (12.5%) M. abscessus sub. abscessus, 2 (8.3%) M. avium sub. avium and 2 (8.3%) were in the M. fortuitum group. Other species included; 1(4.2%) M. bollettii), 1 (4.2%) M. kansasii, 1(4.2%) M. simiae and 1 (4.2%) M. szulgai. GenoType® NTM-DR was performed on MAC and MABC (10 (67%) isolates) of the identified species. Mutations were detected in three isolates of MABC on the erm (41) gene. Two of them are M. abscessus sub. abscessus and one M. abscessus sub. bollettii both with negative band C and positive T band making the isolates resistant to the macrolides. On the other hand no mutation was detected in the rrl and rrs genes hence all MAC were susceptible to both macrolides and aminoglycosides. This study makes it the first attempt to determine the level of awareness among HCW in TB clinics on NTM-PD. It provides baseline information on the level of awareness. It is also the first study in Tanzania to investigate the drug susceptibility profiles of NTM to the recommended drugs. To address the challenges this study uncovers a low HCW awareness on the disease and existence of strains that are resistant to macrolides. Observations predict a low suspicious index (case detection) and treatment failures. In addition to increased burden especially of MAC and MABC, inability to treat with routine TB drugs and under-reporting of cases in Tanzania with no regulated cases management guidelines all these urge TB programs to take action as this has a direct impact on management of TB. The level of awareness on NTM pulmonary disease was generally poor among health care workers at the TB clinics in Northern Tanzania despite of the existence of significant number of cases. Mycobacteria avium complex species have been found the most prevalent NTMs among tested mycobacterial isolates. Finally this study uncovered existence of macrolides resistant mutations among MABC strains. The TB programs should consider building awareness of its team HCWs so can be able to identify cases. As this study reveals an influence of previous participant training to awareness of NTM-PD, we recommend TB programs include a topic on NTM as a module in their current TB training packages. In management of TB, it is important for clinicians to rule out NTM (specifically MAC and MABC) for all patients presenting with TB symptoms. Clinicians should anticipate macrolides resistances especially among MABC infected individuals.