Background Mesoamerica is one of the world's most complex biogeographical regions, mostly due to its complex geological history. This complexity has led to interesting biogeographical processes that have resulted in the current diversity and distribution of fauna in the region. The fish genus Astyanax represents a useful model to assess biogeographical hypotheses due to it being one of the most diverse and widely distributed freshwater fish species in the New World. We used mitochondrial and nuclear DNA to evaluate phylogenetic relationships within the genus in Mesoamerica, and to develop historical biogeographical hypotheses to explain its current distribution. Results Analysis of the entire mitochondrial cytochrome b (Cytb) gene in 208 individuals from 147 localities and of a subset of individuals for three mitochondrial genes (Cytb, 16 S, and COI) and a single nuclear gene (RAG1) yielded similar topologies, recovering six major groups with significant phylogeographic structure. Populations from North America and Upper Central America formed a monophyletic group, while Middle Central America showed evidence of rapid radiation with incompletely resolved relationships. Lower Central America lineages showed a fragmented structure, with geographically restricted taxa showing high levels of molecular divergence. All Bramocharax samples grouped with their sympatric Astyanax lineages (in some cases even with allopatric Astyanax populations), with less than 1% divergence between them. These results suggest a homoplasic nature to the trophic specializations associated with Bramocharax ecomorphs, which seem to have arisen independently in different Astyanax lineages. We observed higher taxonomic diversity compared to previous phylogenetic studies of the Astyanax genus. Colonization of Mesoamerica by Astyanax before the final closure of the Isthmus of Panama (3.3 Mya) explains the deep level of divergence detected in Lower Central America. The colonization of Upper Mesoamerica apparently occurred by two independent routes, with lineage turnover over a large part of the region. Conclusion Our results support multiple, independent origins of morphological traits in Astyanax, whereby the morphotype associated with Bramocharax represents a recurrent trophic adaptation. Molecular clock estimates indicate that Astyanax was present in Mesoamerica during the Miocene (~8 Mya), which implies the existence of an incipient land-bridge connecting South America and Central America before the final closure of the Isthmus of Panama (~3.3 Mya).