Human immunodeficiency virus 1 (HIV-1) is common among injecting drug users (IDUs), and HIV-1 positive IDUs may have potential of HIV transmission among general population through different ways. Therefore, an understanding towards current status of HIV prevalence and genomic characteristics of circulating strains is crucial to devise and implement necessary interventions to control disease in developing countries like Pakistan. A total of 201 plasma samples were collected from IDUs. Samples were first screened using HIV-1 Ag/Ab Combo test and then antigen positive samples were amplified for HIV-1 pol gene (1084 bp) and analyzed. Initial screening showed a total of 49 HIV samples positive (24.37%; 95% CI: 18.96-30.76). A substantial association of HIV incidence was observed in individuals with HCV infection (36.84%; 95% CI: 28.55-45.99; p<0.0001) followed by individuals involved in practices of shared injection equipment (21.17%; 95% CI: 25.07-40.21; p<0.0001), injected previously used syringes (30.2%; 95% CI: 23.4-37.99; p=0.0016), sex with IDUs (37.78%; 95% CI: 25.11-52.37; p=0.002) and those with an age between 30-39 years (27.17%; 95% CI: 19.13-37.04; p=0.039). The pol gene-based phylogeny and subtyping classification categorized the under-study sequences representing subtype A (n = 12; 46.15%), CRF02_AG (n = 6; 23.08%), subtype C (n= 5; 19.23%) and subtype G (n = 3; 11.54%). Subsequent to genotyping resistance interpretation algorithm, one major (M46L) and two accessory (N88D, L89V) PIs mutations in the protease region while four NRTI (D67T, K70R/Q, M184V and T215F) and four NNRTI (V108T, E138A, V179I and Y181C) mutations in the reverse transcriptase region were observed. The present study concludes circulation of multiple subtypes of HIV-1 among IDUs and a continuous disease surveillance coupled with delineation of disease risk factors may provide a crucial insight into HIV prevention and treatment which could substantially curtail HIV epidemics in IDUs.