Background: Tuberculosis (TB) is still one of the major causes of death from a single infectious agent worldwide. The emergence of drug resistance has magnified the problem Aim: To characterize susceptibility of Mycobacterium tuberculosis isolates using novel drug testing and molecular techniques in Moshi Kilimanjaro Tanzania. Methodology: TB isolates (archived vials) of patients confirmed to have TB and have not started medication from KCMC TB laboratory were recultured into LJ media. Culture from LJ media was used to perform molecular speciation for confirmation of the MTB species and drug susceptibility testing of MTBc using MGIT 960(SIRE), TREK/MYCOTB plate, D29 Phage assay, and identification of non-tuberculosis mycobacterium by DNA sequencing. Results: A total of 33 isolates were subjected to molecular speciation. Twenty seven (81.8%) were MTBc and six (18.2%) were NTM. Four NTM (66.7%) out of six were subjected to DNA sequencing. Two (50%) were found to be M.fortuitum, one (25%) M. abscessus and one (25%) M. intracellulare. MGIT 960 was able to identify 13(48.1%) of MDR while TREK/MYCOTB plate identified 12(44.4%) isolates and there were no XDR isolates identified by this method. Conclusion: This study aimed at characterization susceptibility of Mycobacterium tuberculosis using novel drug testing and molecular methods in Kilimanjaro Moshi Tanzania. We were able to find that majority (75%) of the isolates were resistant at least to one first line drug. Almost half were MDR and there were no XDR. Eighteen percent were NTM. Phage assay is a promising susceptibility testing method on MTB. Recommendation: The use of molecular methods to identify MTBc in our setting is important in order to clearly the true pathogen. Also use of Novel drug susceptibility testing for MTB is critically needed in order to improve MDR management. Key words: Tuberculosis, D29 Mycobacteriophage, Phage real time PCR, Monoresistance, Polyresistance, Multi-drug resistance, And drug susceptibility test extensively drug resistance tuberculosis, Minimum inhibition concentration.