Cytotoxic T lymphocyte (CTL) responses are thought to be essential for the control of HIV-1 replication in vivo and immunogens that elicit CTL responses are currently a major focus of HIV vaccine research. Here we investigated two aspects of the CTL response to HIV-1 subtype C that are important for vaccine design and efficacy monitoring. First, we assessed the relationship between the CTL response and sequence diversity, using a robust statistical method. While peptides that were most frequently recognized by the CTL response in Nef and p24 tended to be conserved, this was not the case for p17 where epitope recognition coincided with highly variable regions. Second, we investigated the relationship between observed and predicted CTL responses, given the HLA genotype of infected individuals. Only 52% of the Nef peptides and 64% of the Gag peptides that elicited a CTL response contained sequence motifs thought to be required for binding by the HLA-A or -B alleles found in the corresponding patient. In a comparable subtype B dataset a much higher proportion of the peptides that elicited a CTL response were consistent with the patient HLA genotype (96% and 83% for Nef and Gag, respectively). We demonstrate that this difference between subtypes C and B is likely to result from a combination of a tendency for HLA alleles common in Southern African populations to be poorly characterized, as well as a tendency for sequence motifs associated with HLA recognition to be overspecified for sequence variation found in the B clade. Our results suggest that knowledge of HLA binding motifs is likely to be biased toward certain populations and subtypes. This can have important implications for understanding immune escape and predicting vaccine efficacy in the context of populations primarily infected with non-B subtype HIV-1.