Human immunodeficiency virus type 1 (HIV-1) subtype C is responsible for more than 55% of HIV-1 infections worldwide. When this subtype first emerged is unknown. We have analyzed all available gag (p17 and p24) and env (C2-V3) subtype C sequences with known sampling dates, which ranged from 1983 to 2000. The majority of these sequences come from the Karonga District in Malawi and include some of the earliest known subtype C sequences. Linear regression analyses of sequence divergence estimates (with four different approaches)were plotted against sample year to estimate the year in which there was zero divergence from the reconstructed ancestral sequence. Here we suggest that the most recent common ancestor of subtype C appeared in the mid- to late 1960s. Sensitivity analyses, by which possible biases due to oversampling from one district were explored, gave very similar estimates.