A cross-sectional study was undertaken among drug-naïve HIV patients at the University Hospital in Ouagadougou shortly before and after the introduction of large-scale antiretroviral therapy (ART) in Burkina Faso. Baseline clinical and virological data as well as protease (PR) and 5' reverse transcriptase (RT) sequences from 104 HIV infected patients were analyzed. Genotypic classification revealed the following subtypes and recombinant forms: CRF06_cpx, n = 46 (44.2%); CRF02_AG, n = 39 (37.5%); subtype A, n = 4 (3.8%); CRF09_cpx, n = 2 (1.9%); and unclassified, n = 13 (12.5%). Bootstrap analysis of CRF02_AG and CRF06_cpx viruses showed that >80% had a similar structure to their respective prototypes. The prevalence of primary drug resistance mutations was 12.5%, all mutations arising in the RT sequences in accordance with the dominance of this drug class in Burkina Faso. The mutations were distributed as follows: NRTI (10.6%): M41L (n = 2), D67N (n = 2), K70K/E (n = 2), L210W (n = 1), T215S/Y (n = 2), and K219K/Q (n = 2); NNRTI (6.1%): K103K/N (n = 2), Y181C (n = 2), G190G/A (n = 1), and P236P/L (n = 1). Subtype specific secondary polymorphisms such as K20I and M36I in the PR were observed in almost all patients. Drug resistance mutations occurred at similar frequencies (12.8% and 10.8%, respectively) among patients infected with CRF02_AG and CRF06_cpx. Some subtype specific polymorphisms were observed within important HLA epitopes, including B35, B7, and A2 in the RT, and A*6802 in the PR sequences. The observed resistance mutations are most likely to have been transmitted based on the timing of the study but prior undocumented use of ART cannot be excluded.