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Hylocereus (Berger) Britton and Rose is a cacti with high economic potential. It is distributed in tropical, subtropical and semi-arid environments. The number of species identified in Mexico has been controversial, mainly because of the wide morphological variation observed on their vegetative structures. Three chloroplast regions (matK, rbcL, psbA), and the intergenic spacer trnL-F were sequenced to evaluate their suitability as barcode for H. ocamponis (Salm-Dick) Britton and Rose, H. purpusii (Weingart) Britton and Rose and H. undatus (Haworth) Britton and Rose. The samples were collected from 18 localities and previously characterized based on morphological and anatomical traits. Two species of Selenicereus were used as comparison group. Individual markers rbcL and triiL-F confirmed the differentiation of the three species. The multilocus analysis based on Bayesian inference with rbcL and trnL-F combined also discriminated the three species with probabilities > 60 %. These results suggest that this loci combination could be useful as a barcode for Hylocereus species.