Phylogeography can improve the understanding of local and worldwide HIV epidemics, including the migration of subepidemics across national borders. We analyzed HIV-1 sequences sampled from Mexico and San Diego, California to determine the relatedness of these epidemics. We sampled the HIV epidemics in (1) Mexico by downloading all publicly available HIV-1 pol sequences from antiretroviral-naive individuals in GenBank (n = 100) and generating similar sequences from cohorts of injection drug users and female sex workers in Tijuana, Mexico (n = 27) and (2) in San Diego, California by pol sequencing well-characterized primary (n = 395) and chronic (n = 267) HIV infection cohorts. Estimates of population structure (F(ST)), genetic distance cluster analysis, and a cladistic measure of migration events (Slatkin-Maddison test) were used to assess the relatedness of the epidemics. Both a test of population differentiation (F(ST) = 0.06; p < 0.01) and a cladistic estimate of migration events (84 migrations, p < 0.01) indicated that the Tijuana and San Diego epidemics were not freely mixing. A conservative cluster analysis identified 72 clusters (two or more sequences), with two clusters containing both Mexican and San Diego sequences (permutation p < 0.01). Analysis of this very large dataset of HIV-1 sequences suggested that the HIV-1 epidemics in San Diego, California and Tijuana, Mexico are distinct. Larger epidemiological studies are needed to quantify the magnitude and associations of cross-border mixing.