The assignment of accurate species names is crucial, especially for those with confirmed agronomic potential such as highland papayas. The use of additional methodologies and data sets is recommended to establish well-supported boundaries among species of Vasconcellea. Accordingly, six chloroplast (trnL-trnF, rpl20-rps12, psbA-trnH intergenic spacers, matK and rbcL genes) and nuclear (ITS) markers were used to delimit species in the genus Vasconcellea using phylogeny and four DNA-based methods. Our results demonstrated congruence among different methodologies applied in this integrative study (i.e., morphology, multilocus phylogeny, genetic distance, coalescence methods). Genetic distance (ABGD, SPN), a coalescence method (BPP), and the multilocus phylogeny supported 22–25 different species of Vasconcellea, including the following five new species from northern Peru: V. badilloi sp. nov., V. carvalhoae sp. nov., V. chachapoyensis sp. nov., V. pentalobis sp. nov., and V. peruviensis sp. nov. Genetic markers that gave better resolution for distinguishing species were ITS and trnL-trnF. Phylogenetic diversity and DNA-species delimitation methods could be used to discover taxa within traditionally defined species.