To provide the hard-won information on HIV-1 polymorphism from inmates in prison and to study the genetic variation of HIV-1 strains, we analyzed the characterization of the nucleotide and corresponding protein sequences of naturally occurring nef sequences derived from 16 HIV-1-positive inmates from prisons in China. Nested PCR and DNA sequencing were used to determine nef sequences in peripheral blood mononuclear cells. Phylogenetic analysis revealed a distribution of the viral sequences among subtype B, CRF_BC, and CRF_AE, with subtype B accounting for more than half of the genotypes. When compared with the consensus sequence, a certain amino acid sequence variability was observed. However, most of the described nef functional motifs were relatively well conserved in the majority of the sequences analyzed. Our results indicate that HIV-1 strains found in our study subjects may have a common origin and the description of motifs will produce important information for further studies of nef function.