The agaves are plants of cultural importance which have been used by humans for about 10,000 years and about 40 specific uses. The most culturally and economically important of those uses are for the production of fermented (pulque) and distilled beverages (mescal). Pulque continues to be produced in nearly all of Mexico, and the agaves used for this purpose have shown domestication syndrome. 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 the state of Hidalgo. We did semi-structured interviews, free listings, and tours with 11 agave managers. We analyzed morphology and studied genetic diversity and structure using nuclear microsatellites. We found wild-collected, tolerated, transplanted, and cultivated varieties of agave. This comprised 19 traditional varieties of pulque agave, 12 of them in production during the study, which corresponded to the species Agave americana, A. salmiana y A. mapisaga and five intraspecific entities. The varieties were grouped morphologically according to a management gradient; the wild-collected 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 from 0.204 to 0.721. Bayesian clustering suggested the existence of three genetic groups, both at the level of traditional varieties of pulque agaves and for management categories, a result that matches multivariate clustering. 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-growing agaves, which seems to be due to vegetative propagation, among other factors.