To analyze HIV-1 subtype distribution, sequence analysis was performed on serum specimens obtained in 1994 from the Rakai Health Sciences community cohort in Uganda. Portions of gag-p24 and env-gp41 were sequenced and HIV subtype was determined for 773 subjects residing in 10 community clusters in rural Uganda. Subtypes A (17%) and D (70%) were the most common strains in the population. Subtype distribution varied by geographic region with significantly more subtype A in northern community clusters compared with southern clusters (21% vs. 8%, p < 0.001) and more subtype D in southern clusters compared with northern clusters (78% vs. 65%, p < 0.008). These data illustrate the geographic complexity of subtype variation, which has important implications for HIV-1 vaccine design.