Anopheles halophylus Silva-do-Nascimento & Lourenc¸o-de-Oliveira was recently described using morphological and biological variants in specimens previously identiÞed as Anopheles triannulatus (Neiva & Pinto). Because these two species occur in sympatry in central Brazil, we used allozymes to determine the extent of gene ßow to conÞrm that they are different species. Of 11 allozyme loci analyzed, one (Mpi) was found to be diagnostic for An. halophylus and An. triannulatus, conÞrming their speciÞc status. This locus revealed a second sibling species within An. triannulatus sensu lato. An. halophylus and the new undescribed species were conÞrmed using random ampliÞed polymorphic DNA markers that showed moderate genetic divergence among these three sympatric and closely related taxa (D 0.145Ð0.428). Moreover, this marker indicates that An. halophylus and the new species are more closely related to each other than either is to An. triannulatus.