Physalaemus pustulosus, a small leptodactylid frog with South American affinities, ranges across northern South America through Middle America to southern Mexico. To investigate its geographic variation and evolutionary origins, we analysed the presumptive gene products of 14 allozyme loci and sequenced a portion of the mitochondrial COI gene from individuals sampled throughout the distribution. Generally, allozyme dissimilarities and sequence divergences are correlated with each other and with geographic proximity. The greatest discontinuity in genetic variation was found between populations in Middle America vs. South America + Panama. Based on two Bayesian MCMC (Markov chain Monte Carlo) divergence time estimates involving two independent temporal constraints, the timing of the separation of northern and southern túngara frog lineages is significantly older than the time since completion of the current Panama land bridge. P. pustulosus first invaded Middle America from South America about 6–10 million years ago giving rise to the northern lineage. The southern lineage then invaded Panama independently after land bridge completion. Despite millions of years of independent evolution, the multilocus allozyme data revealed that western Panama populations represent a contact zone containing individuals with alleles from both groups present.