To understand the molecular epidemiology of HIV-1 infection in Iran, we conducted the first study to analyze the genome sequence of Iranian HIV-1 isolates. For this cross-sectional study, we enrolled 10 HIV-1-infected individuals associated with injection drug use from Tehran, Shiraz, and Kermanshah. Near full-length genome sequences obtained from their plasma samples were used for phylogenetic tree and similarity plotting analyses. Among 10 isolates, nine were clearly identified as CRF35_AD and the remaining one as CRF01_AE. Interestingly, five of our Iranian CRF35_AD isolates made two clusters with 10 Afghan CRF35_AD isolates in a phylogenetic tree, indicating epidemiological connections among injection drug users in Iran and Afghanistan. In contrast, our CRF01_AE isolate had no genetic relationship with any other CRF01_AE isolates worldwide, even from Afghanistan. This study provides the first genomic evidence of HIV-1 CRF35_AD predominance and CRF01_AE infection among individuals associated with injection drug use in Iran.