Baseline HIV-1 resistance data are important for resistance monitoring purposes especially in regions initiating large-scale antiretroviral treatment programs. We examined 40 protease and 35 reverse transcriptase amino acid sequences of HIV-1 subtype C from drug inexperienced patients from rural settings in South Africa for resistance mutations. Samples were collected between 2001 and 2004 prior to the availability of antiretrovirals through public health institutions. Ninety-five percent of patients had no major mutations in the protease gene, although substitutions M46L (2.5%) and G73S (2.5%), which according to the Stanford Genotypic Resistance Interpretation Algorithm are considered major mutations, were detected. In addition, a high prevalence of minor mutations was observed in the protease, with at least three minor resistance-associated mutations in 37% of the isolates. An isoleucine insertion at codon 37 was detected in one sequence. Most of the RT sequences were wild-type, although V118I (8.5%) and Y318F (5.7%) associated with resistance to lamivudine and nevirapine, respectively, were observed. Our data suggest that major resistance mutations among the drug-inexperienced population in South Africa may be rare, and routine resistance testing before the initiation of therapy in this initial stage of the treatment program may not be necessary. © Mary Ann Liebert, Inc.