It is known that overlapping HIV-1 peptides of different lengths can be presented by a given HLA class I molecule. However, the role of those peptides in CD8(+) T cells recognition of HIV-1-infected cells remains unclear. Here we investigated the recognition of overlapping 8-mer to 11-mer peptides of Pol 155-165 by HLA-B*54:01-restricted CD8(+) T cells. The analysis of ex vivo T cells using ELISPOT and tetramer binding assays showed that there were different patterns of CD8(+) T-cell responses to these peptides among chronically HIV-1-infected HLA-B*54:01(+) individuals, though the response to the 9-mer peptide was the strongest among them. CD8(+) T-cell clones with TCRs specific for the 9-mer, 10-mer, and/or 11-mer peptides effectively killed HIV-1-infected cells. Together, these results suggest that the 9-mer and 10-mer peptides could be predominantly presented by HLA-B*54:01, though it remains possible that the 11-mer peptide was also presented by this HLA allele. The present study demonstrates effective CD8(+) T-cell recognition of HIV-1-infected cells via presentation of multiple overlapping HIV-1 peptides and cross-recognition by the CD8(+) T cells.