Most HIV-1 transmission studies use self-reported history to define the source contact. To evaluate the reliability of epidemiologic source partner reporting, heteroduplex mobility assays (HMAs) were performed comparing the different viral strains present in the partners. Partners were typed for human leukocyte antigen (HLA) to evaluate the degree of shared alleles. Of 11 couples evaluated, HMA analysis confirmed nine transmissions (including 1 oral-genital transmission), indicated probable transmission in 1 couple, and suggested an alternative source partner in another. Nine source partners transmitted a major variant. Four source partners knew their HIV status. Previous HIV monitoring was reported by 5 of the 6 confirmed source partners who were unaware of their HIV status at the time of transmission. We also evaluated potential “sharing of HLA alleles” as a risk factor for HIV-1 acquisition; partners were not found to have a higher degree of shared HLA alleles. Lack of awareness about infection status as a consequence of infrequent testing plays a major role in the secondary transmission of HIV. These findings re-emphasize the importance of using safe sex practices at all times.