Control of human immunodeficiency virus type 1 (HIV-1) by HLA-B27-positive subjects has been linked to an immunodominant CD8⁺ cytotoxic T-lymphocyte (CTL) response targeting the conserved KK10 epitope (KRWIILGLNK_263-272) in p24/Gag. Viral escape in KK10 typically occurs through development of an R₂₆₄K substitution in conjunction with the upstream compensatory mutation S₁₇₃A, and the difficulty of the virus to escape from the immune response against the KK10 epitope until late in infection has been associated with slower clinical progression. Rare alternative escape mutations at R₂₆₄ have been observed, but factors dictating the preferential selection of R₂₆₄K remain unclear. Here we illustrate that while all observed R₂₆₄ mutations (K, G, Q, and T) reduced peptide binding to HLA-B27 and impaired viral replication, the replicative defects of the alternative mutants were actually less pronounced than those for R₂₆₄K. Importantly, however, none of these mutants replicated as well as an R₂₆₄K variant containing the compensatory mutation S₁₇₃A. In assessing the combined effects of viral replication and CTL escape using an in vitro coculture assay, we further observed that the compensated R₂₆₄K mutant also displayed the highest replication capacity in the presence of KK10-specific CTLs. Comparisons of codon usage for the respective variants indicated that generation of the R₂₆₄K mutation may also be favored due to a G-to-A bias in nucleotide substitutions during HIV-1 replication. Together, these data suggest that the preference for R₂₆₄K is due primarily to the ability of the S₁₇₃A-compensated virus to replicate better than alternative variants in the presence of CTLs, suggesting that viral fitness is a key contributor for the selection of immune escape variants.