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Abstract Aim In this study, we investigate phylogeographic structure in two different species groups of lowland toads. First, we further investigate strict parapatry of the Pliocene-vicariant Bufo valliceps/B. nebulifer species pair. Secondly, we test for similar phylogeographic structure in the distantly related toad B. marinus, a species we hypothesize will show a Pleistocene dispersal across the same area. Location The eastern extension of the Trans-Mexican Neovolcanic Belt (TMNB) contacts the Atlantic Coast in central Veracruz, Mexico. Although it is not a massive structure at this eastern terminus, the TMNB has nonetheless effected vicariance and subsequent speciation in several groups of animals. The Isthmus of Tehuantepec unites the North American continent with Nuclear Central America and is also known to be a biogeographic barrier for many taxa. Methods We use sequence data from two mitochondrial DNA genes (c. 550 base-pairs (bp) of 16S and c. 420 bp of cyt b) from 58 individuals of the B. valliceps/nebulifer complex, collected from 24 localities. We also present homologous sequence data from 23 individuals of B. marinus, collected from 12 localities. We conduct maximum-parsimony, maximum-likelihood and Bayesian analyses to investigate phylogeographic structure. We then use parsimony- and likelihood-based topology tests to assess alternative phylogenetic hypotheses and use a previously calibrated molecular rate of evolution to estimate dates of divergence. Results Our results further define the parapatric contact zone across the TMNB between the Pliocene-vicariant sister species B. valliceps and B. nebulifer. In contrast, phylogenetic structure among populations of B. marinus across the TMNB is much shallower, suggesting a more recent Pleistocene dispersal in this species. In addition, we found phylogeographic structure associated with the Isthmus of Tehuantepec in both species groups. Main conclusions The existence of a Pliocene–Pleistocene seaway across the Isthmus of Tehuantepec has been controversial. Our data depict clades on either side of the isthmus within two distinct species (B. valliceps and B. marinus), although none of the clades associated with the isthmus, for either species, are reciprocally monophyletic. In the B. valliceps/B. nebulifer complex, the TMNB separation appears to predate the isthmian break, whereas in B. marinus dispersal across the TMNB has occurred subsequent to the presence of a barrier at the Isthmus of Tehuantepec.