- Interest in vine cacti of the genus Hylocereus (Cactaceae) has grown markedly due to their high economic potential as exotic fruit crops. Thus, we investigate the genomic and genetic characteristics of 18 accessions belonging to three Hylocereus species, from which were produced eight progeny from self-pollination and 51 interspecific-homoploid and -interploid hybrids. We reported ploidy estimation, allele frequencies, polymorphic information content (PIC) and genetic relationships observed among the Hylocereus species and their progeny. The progeny were diploid, triploid, tetraploid, pentaploid, or hexaploid. Each primer combination used in this work amplified different sets of restriction fragments ranging from 74 to 102 bands. Among the total number of bands observed for the Hylocereus accessions and their progeny, 97.5 and 98.1 %, respectively, were polymorphic. The variability of PIC between primers, species and hybrids suggested high heterozygosity and gene flow between them. In addition, amplified fragment length polymorphism (AFLP) markers were used to successfully identify one of the H. megalanthus accessions beforehand as the unknown male progenitor of the allotriploid S-75. AFLP markers demonstrate the efficacy for assessing genetic relationships and introgression; and provide strong support for the pursuit of additional breeding programs of these fruit crop.