Rhodnius prolixus is one of the most important primary vectors of human Chagas disease in Latin America. Its morphology is, however, identical to that of the members of the Rhodnius robustus cryptic species complex, which includes secondary vectors. The correct identification of these taxa with differential vector competence is, therefore, of great epidemiological relevance. We used the alignment of 26 mitochondrial cytochrome b haplotypes (663 bp) to select for PCR-amplifiable species-specific regions. We designed one forward primer on a region conserved across all haplotypes, and three reverse primers that anneal to species-specific regions and amplify fragments of different lengths for R. prolixus (285 bp) and for members of the two major R. robustus lineages: group I (349 bp) and groups II-IV (239 bp). These fragments were easily identifiable on regular 1.5% agarose gels. This multiplex PCR assay was successfully tested on 81 specimens from six Latin American countries, and used to determine the phylogeographic boundaries for each species. It is a simple, objective, and cost-effective assay. Its PCR-based nature makes it applicable to any insect developmental stage, as well as to dried specimens, and insect remains. It should be particularly useful in areas where representatives of these Rhodnius species occur in sympatry.