Prevention of mother-to-child transmission (PMTCT) guidelines recommend that all HIV-infected pregnant woman receive antiretroviral therapy (Option B) and HIV-infected infants should initiate therapy with a protease inhibitor based regimen; however, implementation of these guidelines has lagged in many resource-limited settings. Tanzania only recently implemented these guidelines with little country specific data to inform whether HIV non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance was present among infected infants under the Option A guidelines. This study aimed to identify primary resistance mutations in HIV-infected infants and to identify risk of NVP resistance based on maternal and infant NVP exposure. Infant dried blood spots were sent to the zonal reference laboratory at Kilimanjaro Christian Medical Centre (KCMC) Clinical Laboratory and underwent DNA PCR testing for HIV as standard of care. Using the Clinical Laboratory registry, HIV-positive DBS cards, stored at ambient temperature, were identified and sent for further viral load testing, nucleotide sequencing and analysis. Clinical information was obtained from the PMTCT clinical sites and the National PMTCT registry for information regarding maternal and infant demographics and PMTCT treatment regimen. Results demonstrated that infants exposed to NVP were more likely to have high level resistance mutations to NVP as compared to those infants not exposed to NVP (p=0.002). The most common high-level resistance mutations to NVP were K103N, Y181C, and Y188L. HIV subtype A was most common, followed by subtype C. Approximately one-third of HIV-infected infants had documented referral to HIV care. This study demonstrated the ongoing need to scale up and strengthen points along the PMTCT continuum and supported the recommendation for all HIV-infected infants to initiate a lopinavir/ritonavir-based antiretroviral therapy regimen.