We examined strontium isotope ratios (87Sr/86Sr) in fish otoliths to determine the origins of invasive piscivores in the Upper Colorado River Basin (UCRB, western USA). We examined87Sr/86Sr from fishes in different reservoirs, as well as the temporal stability and interspecies variability of87Sr/86Sr of fishes within reservoirs, determined if87Sr/86Sr would be useful for “fingerprinting” reservoirs where invasive piscivores may have been escaping into riverine habitat of endangered fishes in the UCRB, and looked for evidence that such movement was occurring. Our results showed that in most cases87Sr/86Sr was unique among reservoirs, overlapped among species in a given reservoir, and was temporally stable across years. We identified the likely reservoir of origin of river-caught fish in some cases, and we were also able to determine the year of possible escapement. The approach allowed us to precisely describe the87Sr/86Sr fingerprint of reservoir fishes, trace likely origins of immigrant river fish, and exclude potential sources, enabling managers to focus control efforts more efficiently. Our results demonstrate the potential utility of87Sr/86Sr as a site-specific and temporally stable marker for reservoir fish and its promise for tracking fish movements of invasive fishes in river–reservoir systems.