This study analyzes the phylogenetic and phylogeographic relationships of the rattlesnake, Crotalus durissus, throughout its geographical range using several molecular markers. The Cyt b and ND4 fragments of the mtDNA were sequenced and analyzed. The results support the monophyly of all South American C. durissus populations, including populations that had previously been considered as separate species. The Cyt-b and ND4 analysis indicates that the Central American and Mexican C. durissus populations are paraphyletic. The nuclear intron 7 of ~-fibrinogen was also studied. However, it did not aid in resolving the phylogenetic relationships of the populations analyzed. Amplified fragment length polymorphism (AFLP) analysis suggested phylogenetic patterns similar to those indicated by mtDNA analysis, except for small inconsistencies in the topologies. Pairwise and partial Mantel tests showed a significant correlation between AFLP and mtDNA phylogenies, taking into consideration the geographic distance effect. This study reveals that C. durissus recently dispersed into South America. The timing of the dispersal event is consistent with the hypothesis that this species invaded South American 1-3 Mya after the uplift of the Panama land bridge. The Central American lineages are much older and divergent, the products of orogenic evolution in Mexico. The implications for systematics include a reconsideration of the status of some taxa (C. unicolor and C. vegrandis) that clearly appear conspecific of South American C. durissus. The Mexican lineages are regarded as full evolutionary species. The phylogeography of C. durissus in South America shows a stepwise colonisation progressing from a northern centre of origin in Mexico to northern South America, and across the Amazon Basin. The pattern consists of a set of nested clades, in which any southern clade is nested within a paraphyletic group consisting of more no1thernly haplotype clades. Low sequence divergence between populations from north and south of the Amazon rainforest is consistent with mid-Pleistocene divergence, approximately 1.08 million years ago. This suggests that the Amazonian rainforests must have become fragmented or considerably constricted during that period.