Specimens of neotropical Anopheles (Nyssorhynchus) were collected and identified morphologically. We amplified three genes for phylogenetic analysis-the single copy nuclear white and CAD genes, and the COI barcode region. Since we had multiple specimens for most species we were able to test how well the single or combined genes were able to corroborate morphologically defined species by placing the species into exclusive groups. We found that single genes, including the COI barcode region, were poor at confirming species, but that the three genes combined were able to do so much better. This has implications for species identification, species delimitation, and species discovery, and we caution that single genes are not enough. Higher level groupings were partially resolved with some well-supported groupings, whereas others were found to be either polyphyletic or paraphyletic. There were examples of known groups, such as the Myzorhynchella Section, which were poorly supported with single genes but were well supported with combined genes. From this we can infer that more sequence data will be needed in order to show more higher-level groupings with good support. We got unambiguously good support (0.94-1.0 Bayesian posterior probability) from all DNA-based analyses for a grouping of An. dunhami with An. nuneztovari and An. goeldii, and because of this and because of morphological similarities we propose that An. dunhami be included in the Nuneztovari Complex. We obtained phylogenetic corroboration for new species which had been recognised by morphological differences; these will need to be formally described and named.