Background: Mother-to-child transmission (MTCT) of HIV remains an important problem in Sub-Saharan Africa (SSA) where most new pediatric HIV infections occur. EID of HIV provides an opportunity to assess current MTCT rates. Furthermore, effectiveness of scale-up in PMTCT programs can be assessed. Objective: This study intended to determine five years prevalence of HIV among infants born to HIV-infected mothers and the progress in prevention of MTCT corresponding to scale-up of PMTCT programs in Northern Tanzania. The progress in utilization of DBS PCR testing of HIV for infants was also investigated retrospectively. Methodology: Retrospective cross-sectional study. Five-year records of 18934 HIV-exposed infants from all PMTCT programs in four regions (Arusha, Kilimanjaro, Manyara and Tanga) of Northern Tanzania were collected from the Laboratory Information System (LIS), the DISA system of the Zonal PCR laboratory. These data were assessed to determine MTCT of HIV trend from 2011-2015. Data were entered, summarized and analyzed using SPSS for windows version 22.0 (2013) Armonk, NY, USA. Results: Of 18873 evaluable infants in the study, 970 had a positive DBS PCR result at ≤ 75 days of life, for an overall prevalence of 5.1%. Prevalence decreased substantially after rollout of WHO Option B+, but it went high in 2015. The overall prevalence for individual regions were essentially approaching each other with Arusha top in the list for 5.4% HIV prevalence among exposed infants. Uptake of DBS PCR for EID was promisingly high for the whole study period. Conclusion: The study demonstrated overall 5.1% prevalence of HIV among exposed infants in four regions of Northern Tanzania. Reductions in HIV prevalence among exposed infants have been observed following WHO Option B+, and the uptake of DBS PCR for EID is encouraging.