The development of resistance mutations in drug-targeted HIV-1 genes compromises the success of antiretroviral therapy (ART) programs. Genotyping of these mutations enables adjusted therapeutic decisions both at the individual and population level. We investigated over time the prevalence of HIV-1 primary drug resistance mutations in treatment-naive patients and described the HIV-1 subtype distribution in a cohort in rural Tanzania at the beginning of the ART rollout in 2005-2007 and later in 2009. Viral RNA was analyzed in 387 baseline plasma samples from treatment-naive patients over a period of 5 years. The reverse transcriptase (RT) and protease genes were reversely transcribed, polymerase chain reaction (PCR) amplified, and directly sequenced to identify HIV-1 subtypes and single nucleotide polymorphisms associated with drug resistance (DR-SNPs). The prevalence of major DR-SNPs in 2005-2007 in the RT gene was determined: K103N (5.0%), Y181C (2.5%), M184V (2.5%), and G190A (1.7%), and M41L, K65KR, K70KR, and L74LV (0.8%). In samples from 2009 only K103N (3.3%), M184V, and T215FY (0.8%) were detected. Initial frequencies of subtypes C, A, D, and recombinants were 43%, 32%, 18%, and 7%, respectively. Later similar frequencies were found except for the recombinants, which were found twice as often (15%), highlighting the subtype diversity and a relatively stable subtype frequency in the area. DR-SNPs were found at initiation of the cohort despite very low previous ART use in the area. Statistically, frequencies of major mutations did not change significantly over the studied 5-year interval. These mutations could reflect primary resistances and may indicate a possible risk for treatment failure.