Background: The agaves are plants of cultural importance that have been used by humans for about 10,000 years for about 40 specific uses, of which the most culturally and economically important are fermented (Pulque) and distilled beverages (Mescal). Pulque continues to be produced in nearly all of Mexico, and the agaves used for this propose have shown domestication syndrome and selected morphological characteristics, such as larger plant size (since size is positively related to sap production) and less dentition of the leaves.Methods: In this study, we carry out an ethnobotanical, morphological and genetic analysis of the traditional varieties of pulque agave used in the production of aguamiel (agave sap) and pulque in two localities of the state of Hidalgo. We did semi-structured interviews and tours with crop owners, tlachiqueros (sap harvesters), and people related to the production system. We analyzed the morphology of 111 plants, and we studied the diversity and genetic structure of 130 individuals using 16 nuclear microsatellites.Results: We found wild, tolerated, transplanted, and cultivated varieties of agave. Asexual reproduction was the main method of propagation. We recorded 19 traditional varieties of pulque agave, 12 of them in production during the study, which correspond to the species Agave americana, A. salmiana y A. mapisaga with five intraspecific entities. The varieties were grouped according to a management gradient; the wild varieties were the smallest, with more lateral teeth and a larger terminal spine. The cultivated varieties clearly exhibited domestication syndrome, with larger plants and smaller dentition. The expected heterozygosity (He) of the varieties ranged between 0.204 to 0.721. Bayesian clustering suggested the existence of three genetic groups (K = 3), both at the level of traditional varieties of pulque agaves and for management categories, a result that matches multivariate clustering.Conclusions: Pulque producers in the studied localities, maintain high agrobiodiversity. The cultivated varieties exhibit domestication syndrome, as has been reported for other species of the genus with the same selection purposes. Our results support the hypothesis of a decrease in genetic diversity in crops compared to wild agaves, which seems to be due to vegetative propagation, among other factors.