HIV-1 subtyping is important to study the changing scenario of genetic variation. The gag-based heteroduplex mobility assay (gag-HMA) was developed and evaluated as a powerful and reliable technique for identifying the HIV-1 group M subtypes A to H and the circulating recombinant forms (CRFs). To study the subtype distribution of HIV-1 strains from the eastern part of India, we used the gag-based HMA, followed by the sequencing and phylogenetic analysis. Blood samples from HIV-1-seropositive female sex workers in Calcutta were subjected to gag-HMA. The most prevalent subtype was found to be the C type, among which the C4 subsubtype was prevalent. However, a number of nontypable C strains were found in gag-HMA. Phylogenetic analysis revealed the discrete nature of the C strains and no monophyletic cluster was noticed. This result might indicate a growing tendency of variations among the HIV-1 type C strains circulating in eastern India.