Background: The dramatic scaling up of antiretroviral therapy (ART) prophylaxis in Low and Middle Income Countries (LMIC) among pregnant and breastfeeding women has reduced transmissions among infants born to mothers infected with human immunodeficiency virus (HIV). The use of prophylactic antiretroviral drugs by pregnant women infected by the virus for prevention of mother to child transmission (PMTCT) of HIV-1, may lead to the virus to acquire resistance to antiretroviral drugs. HIV acquired resistance may be transmitted to the children, compromising the future regimens. Limited information is available on the transmitted HIV-1 subtypes and prevalence of mutations associated with antiretroviral drugs among infected children in Tanzania. We characterized transmitted HIV-1subtypes and investigated the prevalence of mutations associated with antiretroviral drugs resistance among children born to seropositive mothers in northern Tanzania. Objective: The aim of the study was to determine transmitted HIV-1 subtypes and prevalence of mutations associated with antiretroviral drug resistance among children born to mothers enrolled in PMTCT in Northern Tanzania. Materials and Methods : Dried Blood Spots (DBS) were collected among children aged less than 18months born to HIV-1 positive mothers from four regions of northern Tanzania attending post-natal clinics at four weeks and six months after birth from January 2011 to December 2012.The DBS were posted from the sites to Kilimanjaro Christian Medical Center (KCMC) clinical laboratory, which is a zonal laboratory for Northern Tanzania and later shipped to Botswana Harvard HIV Research Laboratory (BHHRL) in Gaborone Botswana for analysis. Results: We successfully generated HIV-1 pol sequences on 46 DBS among 122 DBS tested and detected HIV-1 subtypes A (59 %), C (22%), D (7%), A/D (9%) and CRF10_CD (4%). Major drug resistance associated mutations (DRAMs) were found in 28.7% of the generated sequences and mutations associated with NNRTIs were Y181C (9), K103N (4), Y181Y (1), Y188L (1) and G190A (1), while only one mutation M184V associated with NRTI. None of the major mutations associated with PIs was detected. Conclusion and recommendations: The high prevalence of major HIVDR mutations among children infected with the virus in our study subjects suggests the need for close monitoring and routine surveillance, which will provide information on patterns of HIVDR hence early intervention. The predominance of HIV-1 subtypes A among children born to HIV infected mothers indicates high transmissibility capacity of subtype A or that most of the pregnant women in this population were infected with the subtype. This study included only four regions in Tanzania, the expansion of a similar study to other regions of the country to get the national prevalence of transmitted HIV-1 subtypes and mutations associated with treatment failure among children born to mothers on prophylaxis for PMTCT as well as for development of treatment policies and guidelines.