Envelope coreceptor tropism is a phenotypic characteristic of HIV-1 which may be estimated by genotypic algorithms and confirmed through phenotypic assays using virus isolates or recombinant viruses. Viruses can use either CCR5 (R5) or CXCR4 (X4) to enter host cells. Most studies have focused on subtype B HIV infection, although subtype C HIV-1 currently accounts for about 50% of infections globally. Previously, we identified an unexpectedly high frequency of subtype C X4-using virus among treated patients. The clinical isolates were frequently dual mixed (used both CCR5 and CXCR4) and it was not possible to distinguish between mixed infection with exclusively R5 and X4 virus and dual tropic viruses that could use either receptor (R5/X4). Therefore, we generated biological clones from 18 isolates obtained from 12 patients through limiting dilution and identified X4 and X4R5 viruses. Analysis of the biologic clones demonstrated that X4 and R5/X4 coreceptor tropism is associated with length polymorphisms, net charge and structural features of the V3 loop. Phylogenetic analyses confirm that emerging X4-usage in subtype C arises through the initial outgrowth of viruses that are R5/X4 sharing 99% or more env homology with R5 virus. Single nucleoside changes resulting in an amino acid net charge increase in the V3 loop as well as 2 amino acid insertions accounted for most of the X4 and R5X4 clonal isolates. Further characterizations of subtype C envelope are warranted to implement efficient genotyping algorithms to identify exclusive R5 use and better understand tropism and pathogenesis among Subtype C HIV-1 isolates.