Hylocereus (Berger) Britton and Rose and Selenicereus (Berger) Britton and Rose are two genera of vine cacti, commonly named pitahaya, that produce fruit that are gaining in popularity as an exotic fruit in many countries. There has also been an increasing interest in these fruits because they can be grown in areas that are prone to drought and heat where other fruit production is not possible. However, there is significant taxonomic confusion regarding species within these genera as well as some uncertainty among named varieties. Amplified fragment length polymorphism (AFLP) was used to genotype a large pitahaya collection to determine if there was redundancy in some of the commonly named varieties as well as to assess the overall diversity of the collection. A total of 51 markers were scored from 230 accessions. Seven distinct clades were found with 126 putative clones but only three clades had high bootstrap (greater than 80%) support and one had moderate support (60%). Some of the differently named varieties were identical based on our analysis, but there was also genotypic diversity within putatively named varieties. The results of this study will help growers and researchers to choose genetically distinct accessions from the germplasm collection to investigate how different accessions perform in their growing regions.