Mitochondrial DNA (mtDNA) was characterized for restriction fragment length polymorphism (RFLP) to evaluate the genetic variability of continental and island populations of Bothrops jararaca and the closely related B. alcatraz. Resultant data showed a geographic orientation in the genetic differentiation in B. jararaca and high haplotype similarity of B. alcatraz with the northern and middle latitude assemblage of B. jararaca. In the dendrogram illustrating haplotype divergences, two major clusters, conforming to geographical provenance of the snakes, were recognized. One cluster contained B. jararaca from the northern and intermediary areas and B. alcatraz, while the other cluster was composed only of B. jararaca sampled in temperate regions. MtDNAs of island populations of B. jararaca were very similar to each other as well as to those of the adjacent continental populations This indicates that the isolation periods of these islands were not long enough for their populations to accumulate differential substitutions in the mtDNA fragments.