Abstract Archeological excavations of Amerindian sites on Dos Mosquises Island, Los Roques Archipelago, Venezuela, uncovered a wide range of evidence reflecting seasonal exploitation of local resources and multiple ritual depositions of large quantities of ceramic figurines, lithics, and faunal remains. Zooarchaeological analysis revealed the presence of modified and unmodified bones and teeth from numerous imported mammal species. Local geographic and environmental conditions preclude permanent establishment of terrestrial mammal populations and as such, there are no native mammalian taxa on the island itself or the surrounding oceanic archipelago. The presence of these faunal remains on Dos Mosquises can be attributed to the intentional movement of animal resources from the mainland to Los Roques by indigenous groups in the Late Ceramic Age (~AD 1200–1500). Despite attributions to a mainland source region, little else is known about the origins of these unique specimens. Here, we apply strontium (87Sr/86Sr), oxygen (δ18O), and carbon (δ13C) isotope analyses of tooth enamel from various archeologically recovered taxa including deer, peccary, tapir, ocelot, margay, opossum, fox, and weasel to investigate their geographic origins via comparisons with macro-regional models of precipitation δ18O and bioavailable 87Sr/86Sr. The 87Sr/86Sr results are highly variable both for the overall assemblage and between specimens within the same taxa, indicating origins from different geochemical environments of mainland South America. The combined archeological and isotopic evidence are consistent with origins within the late pre-colonial Valencioid Sphere of Interaction which encompassed the Lake Valencia Basin, surrounding regions, and several offshore island groups including Los Roques archipelago.