Genetic heterogeneity in the nef genes from human immunodeficiency virus type 1 (HIV-1)-infected rapid progressors (RPs) and long-term nonprogressors (LTNPs) was analyzed to identify various amino acid substitutions responsible for the discernible difference in disease progression. It was found that the majority of the strains characterized belonged to subtype C, followed by several BC recombinants and subtype A1. Complete nef subtype C sequences from 33 RPs and seven LTNPs were compared, and it was observed that, in the majority of the sequences from both groups, highly conserved functional motifs showed subtle changes. However, drastic changes were observed in two isolates from LTNPs where the arginine cluster was deleted, while in one of them, additionally, acidic residues were replaced by basic residues (EEEEE→RK(R)KKE). The deletion of the arginine cluster and the mutation of acidic residues to basic residues are predicted to delay disease development by abolishing CD4 downmodulation and causing diminution of major histocompatibility complex class I (MHC-I) downregulation, respectively. Nonetheless, this is an exclusive finding in these LTNPs, which necessitates their analysis at the functional level. The synonymous-to-nonsynonymous substitution ratio was greater than one in both of the groups, suggesting amino acid sequence conservation and functional robustness. Interpatient nucleotide distance within the group and between the two groups showed very little variation, confirming genetic relatedness among isolates.