The Yucatan Peninsula (YP) presents heterogeneous environments in a karstic landscape that has been formed from permeable sedimentary rocks dating from the Cretaceous period. Its aquifers now face significant pressure from tourism, agriculture, soil use changes and population growth. Aquifer delimitation typically relies on environmental and socioeconomic criteria, overlooking the subterranean fauna. Stygobiotic crustaceans are highly diverse in the YP’s subterranean karstic systems, expressing adaptations to extreme environments while often also displaying the primitive morphology of evolutionary relics. With distributions restricted to specific environments, they are potential markers of water reserves. A literature review recovered records of 75 species of crustaceans from 132 subterranean systems in the YP, together with geomorphological, hydrological, hydrogeochemical and historical precipitation data. Fourteen UPGMA clusters were informative for mapping species composition, whereby the “Ring of Cenotes”, “Caribbean Cave” and “Cozumel Island” regions were delineated as consolidated aquifers. These aquifers are distinguished by abiotic factors as well: freshwater species dominate the Ring of Cenotes, while marine-affinity species characterize the Caribbean Cave and Cozumel Island aquifers. Stygobiotic crustaceans, being linked to geologically ancient water reserves and having a restricted distribution, offer a complementary tool for aquifer delimitation. Their presence suggests long-term and stable water availability. The use of these unique organisms for integrative aquifer delimitation can provide a way to improve the monitoring networks of regional aquifers.