The ability of HIV-1 to evolve resistance to antiretroviral drugs leads to treatment failure. By nucleotide sequencing of HIV-1 subtype B isolates, amino acids responsible for drug resistance have been identified. Less information is available, however, on the extent and distribution of these amino acids in HIV-1 nonsubtype B viruses circulating mainly in developing countries. More HIV-infected patients in the developing world are now using antiretroviral drugs, and hence there is a need to monitor drug resistance mutations in HIV-1 non-subtype B viruses. This study examines the prevalence of drug resistance mutations in 28 antiretroviral drug-naive HIV-1-infected Zambians. HIV-1 proviral DNA was extracted from peripheral blood mononuclear cells. The region encompassing gag p17 to env C2-V3-C3 was amplified by the polymerase chain reaction followed by direct sequencing. Sequence analyses for drug resistance-associated mutations in th e protease and reverse transcriptase genes, and HIV-1 subtyping, were done. Overall, 92.8% of the generated sequences were HIV-1 subtype C. The generated sequences revealed only secondary associated, but no primary, drug-resistance mutations The most frequent secondary mutations in the protease and RT genes were, respectively, I93L(91.7%), L89M (79.2%), M3611V (79%, 4.2%), and R211K (70.8%), S48T (62.5%). The atypical residues M41N (3.6%) and D67A (3.6%) were detected in the RT gene. This study reveals many naturally occurring polymorphisms in HIV-1 subtype C isolates from antiretroviral drug-naive individuals. Such polymorphisms could lead to rapid treatment failure and development of drug-resistant HIV-1 mutants in individuals undergoing antiretroviral therapy.