Contradictory results have been reported on the impact of duplications/insertions in the HIV-1 gag-p6 late assembly domains [TSG101-binding P(T/S)APP motif and ALIX-binding LYPxnLxxL motif] heterogeneity following therapy failure. However, most studies are limited to small numbers of patients and do not include samples from South Africa, which has the largest number of HIV-1C-infected patients (HIV-1CZA). In this study we compared the gag-p6 variability among HIV-1CZA-infected patients from a South African clinical cohort who experienced antiretroviral therapy (ART) failure (n = 845) with ART-naive HIV-1CZA sequences (n = 706) downloaded from the Los Alamos database. Partial (PTA/PTV/APP) or complete P(T/S)APP duplications were less frequent in HIV-1CZA with ART failure compared to therapy-naive ones (14% vs. 30%; p < 0.001). In contrast, the tetrapeptide PYxE insertion, recently described by us, occurred more frequently (5-fold) in therapy-failure patients (p < 0.001) and was associated with a higher number of reverse transcriptase inhibitor (RTI) mutations (p = 0.04) among patients failing ART.