The mongoose is the principal reservoir for rabies on the island of Puerto Rico. This report describes a molecular epidemiological study of representative rabies viruses recovered from the island in 1997. Two closely related but distinct variants circulating in regionally localised parts of the island were identified. The lack of a monophyletic relationship of these viruses suggests that two independent incursions of rabies onto the island have occurred. Both of these Puerto Rican variants were closely related to a variant, known as the north central skunk strain, currently circulating in North American skunk populations and all are members of the cosmopolitan rabies lineage spread during the colonial period. However, the Puerto Rican viruses are clearly distinct from those presently circulating in mongooses in Cuba and which are epidemiologically closely linked to the Mexican dog rabies virus. This study clearly establishes the distinct origins of the rabies viruses now circulating on these two Caribbean islands.