Background: Mutation at amino acid position 61 of vpu influences the stability, release and toxicity of the HIV-1. Previously, we have reported that the seryl residue 61 of HIV-1 was mostly conserved among the HIV-1 infected Intravenous Drug Users (IDUs) of Manipur. In this study, we further analyzed the site mutagenesis at seryl residue 61 among the heterosexually transmitted HIV-1 infected individuals of Manipur. Methodology: Heterosexually transmitted HIV-1 infected blood samples from forty eight individuals have been studied by viral amplification and sequencing at vpu gene of HIV-1. Translated amino acid sequences were aligned with reference strain of HIV- 1 for analysis of amino acid variation at position number 61. Phylogenetic tree were also interfered among the studied sample. Results: The results revealed that 91.67% of heterosexually transmitted HIV-1 infected individuals were harboring virus with conserved seryl residue 61 which has viral-enhancement function while only 8.33% harbored mutated virus. Phylogenetic tree of the vpu sequences among the heterosexually transmitted HIV-1 infected individuals of Manipur displayed multiple clusters. Conclusions: Among the heterosexually transmitted HIV-1 infected individuals of Manipur, vpu is highly conserved at amino nacid position 61 with serine (wild-type) which predicts virus to be more virulent. The analysis also indicated that the heterosexually transmitted virus was possibly introduced from multiple sources. The understanding developed on HIV-1 pathogenicity will provide vital information for the policy makers and clinicians for sentinel surveillance of this pandemic and may help the vaccine/ drug developers to design effective anti-HIV-1 therapeutic agents to fight back the deadly AIDS pandemic.