Aim To evaluate the genetic diversity and phylogeographical structure of Boa constrictor imperator, in order to identify the key historical events responsible for its current distribution and diversity.

Location The Neotropical region of Mexico and mainland Central America.

Methods We used data from the mitochondrial cytochrome b gene, nuclear ornithine decarboxylase intron and microsatellites to perform spatial genetic analyses, and coalescence methods to infer phylogeographical structure, divergence times and historical demography.

Results Cytochrome b results revealed two main reciprocally monophyletic lineages, one along the Mexican Pacific coast and another along the Gulf of Mexico, Yucatán Peninsula and Central America, diverging c. 5.2 Ma. Both lineages are subdivided into haplogroups and show steady historical growth and a more recent population expansion. High genetic diversity was observed for both cytochrome b (h = 0.944) and microsatellites (H N ei = 0.810–0.900).