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Objective: To ascertain the role of HIV-1 gp120 env PNGs variations and sequence length polymorphism following transmission events as possible supporting forensic evidence to determine directionality of HIV transmission. Method: An observational study of HIV-1 infected family members, where median and range values of the amino acid lengths and PNGs for the genotyped C2V5 region were calculated. Wilcoxon rank-sum test was used to determine differences in these parameters between different family members. Results: For heterosexual transmission, two mothers had longer C3 sequences relative to that of their spouses; p=0.006 and=0.025 whilst the opposite was observed for one mother, p = 0.028. No clear trends were observed for PNGs. Index children had longer C2V5 amino acid sequences compared to their mothers p = 0.013, 0.040, 0.043 for families 205, 375, 567 respectively. Second siblings “V4 and V5 sequences were generally shorter relative to the maternal ones p = 0.039 and 0.028, respectively. Adults had longer V3 amino acid sequences compared to children; p = 0.018. Similar trends were also observed regarding PNGs within the entire C2V5 region, C3 and V4 sub-regions; p= 0.0025, 0.005 and 0.008, respectively. First siblings’ C2V5 and C3 sequence lengths were significantly longer relative to those of the second siblings; p = 0.005 and 0.007, respectively. Conclusion: Our results are suggestive that HIV-1 env C2V5 amino acid length polymorphism and PNGs tend to increase with age and HIV disease progression. Though sensitive and should be cautiously handled, it is tempting to propose the direc-tionality of the HIV transmission events with respect to C3 sequence length polymorphisms. Correlating HIV-1 env C2V5 amino acid length polymorphism and age of infection may be the first step towards a possible valuable piece of forensic evidence which may be useful in criminalisation of willful HIV infections. However, bigger studies are war-ranted to substantiate the authenticity of this potentially useful application.