HIV-1 C2-V3 subgenomic regions of the env gene from Iranian seropositive injecting drug users (IDUs) living in Mashhad have been analyzed to evaluate molecular and phylogenetic relationships with IDUs living in Tehran and identify possible common founder virus isolates. The results show that the viral sequences of the Iranian IDUs are strongly related and form a single cluster within the A subtype related to African Ugandan/Kenyan sub-Saharan isolates. Pairwise nucleotide alignment shows higher average divergence values within the Mashhad group than the Tehran group. Furthermore, the Mashhad sequences show much less conserved amino acid residues in the V3 loop than the Tehran sequences. These data represent the first macro-analysis of the HIV-1 molecular evolution in the Iran and Middle East epidemics and may be extremely relevant to guide the development and implementation of diagnostic as well as preventive/therapeutic approaches in this region.