Aims: To determine the prevalent subtypes of HIV-1 in serodiscordant couples. Setting: Integrated Counselling and Testing Centre (ICTC), Department of Microbiology. Study Design: Prospective pilot study. Participants: Thirty HIV-1 serodiscordant couples. Inclusion Criteria: a) Documentation of HIV-1 infection in one partner and seronegative status in the other, current history of continued unprotected sexual activity within the partnership, demonstration that they have been in a partnership for at least 1 year and are not currently on highly active antiretroviral therapy HAART; b) willingness of both partners to provide written informed consent including consent to continued couple counselling for 3 months. Materials and Methods: HIV-1 subtyping was carried out by heteroduplex mobility analysis (HMA) by amplifying env region; and DNA sequencing by amplifying gag region. Results: HIV-1 env gene was amplified successfully in 10/30 samples; gag gene, in 25/30 samples; and both env and gag gene were amplified successfully in 5/30 samples. HIV-1 subtype C was detected from 21 samples; subtype B, from 7; and subtype A, from 2. Sample from 1 positive partner was detected as subtype C by env HMA and subtype B by gag sequencing. Conclusion: HIV-1 subtype C was found to be the predominant subtype of HIV-1 in serodiscordant couples attending our ICTC, followed by HIV-1 subtype B and HIV-1 subtype A, respectively. DNA sequencing was found to be the most reliable method for determining the subtypes of HIV-1.