An HIV-1 subtype C specific assay was established for integrase genotyping from 51 integrase inhibitor-naive patient plasma samples and 22 antiretroviral drug-naive primary viral isolates from South Africa. Seventy-one of the 73 samples were classified as HIV-1 subtype C and two samples were unique AC and CG recombinants in integrase. Amino acid sequence analysis revealed there were no primary mutations (Y143R/C/H, Q148H/R/K, and N155H/S) associated with reduced susceptibility to the integrase inhibitors raltegravir and elvitegravir. However, one sample had the T97A mutation, three samples had the E157Q and V165I mutations, and the majority of samples contained the polymorphic mutation V72I. The expected finding of no major integrase mutations conferring resistance to integrase inhibitors suggests that this new antiretroviral drug class will be effective in our region where HIV-1 subtype C predominates. However, the impact of E157Q and other naturally occurring polymorphisms warrants further phenotypic investigation.