Homegardens are reservoirs of genetic resources. When native plant populations are compromised, traditional management of homegardens can be an effective means of conserving species and enhancing food security. Hylocereus undatus (dragonfruit) is a key species in homegardens but is rarely found in wild populations in the Yucatan. We analyzed management practices and genetic diversity of dragonfruit grown in homegardens in three climate regions (West, Central, and East) in the state of Yucatan, Mexico. Dragonfruit presence and reproductive phenology were documented via 2,660 surveys applied in seventeen municipalities. Eighty-nine semi-structured interviews were conducted to document dragonfruit management and use. Seventy-one specimens were collected among nine populations to characterize genetic diversity with DNA Inter Simple Sequence Repeats (ISSRs) markers. Dragonfruit was found in 12.4% of homegardens, propagated via cuttings (98%), received minimal agricultural management, and flowered and fruited in different seasons among regions. Its fruits were consumed fresh or as a beverage and different parts of the plants were used to treat dysentery, kidney stones, hair loss, and gastritis. Genetic diversity among regions was moderate (PLP [polymorphic loci] = 25.1 to 53.3%; H_e [expected heterozygosity] = 0.075 to 0.138), but the polymorphism information content (PIC) values were at the most 20% lower than those estimated from an ex situ germplasm collection of Hylocereus spp. A Bayesian assignment analysis identified two genetic lineages (K = 2): one in the West and East regions and another in the West and Central regions. Genetic differentiation Jaccard indexes among accessions from all homegardens were similar to those found among wild accessions from Mexico. We conclude, therefore, that homegardens in Yucatan are important in conserving the regional biocultural heritage of dragonfruit.