Resource managers are concerned that water conservation practices in irrigated farmlands along the southern border of the Salton Sea, Imperial County, California, could increase selenium concentrations in agricultural drainwater and harm the desert pupfish (Cyprinodon macularius), a federally protected endangered species. As part of a broader attempt to address this concern, we conducted a 3-year investigation to collect baseline information on selenium concentrations in seven agricultural drains inhabited by pupfish. We collected water, sediment, selected aquatic food-chain taxa (particulate organic detritus, filamentous algae, net plankton, and midge [Chironomidae] larvae), and two poeciliid fishes (western mosquitofish Gambusia affinis and sailfin molly Poecilia latipinna) for selenium determinations. The two fish species served as ecological surrogates for pupfish, which we were not permitted to sacrifice. Dissolved selenium ranged from 0.70 to 32.8 μg/L, with selenate as the major constituent. Total selenium concentrations in other environmental matrices varied widely among drains, with one drain (Trifolium 18) exhibiting especially high concentrations in detritus, 5.98–58.0 μg Se/g; midge larvae, 12.7–50.6 μg Se/g; mosquitofish, 13.2–20.2 μg Se/g; and mollies, 12.8–30.4 μg Se/g (all tissue concentrations are based on dry weights). Although toxic thresholds for selenium in fishes from the Salton Sea are still poorly understood, available evidence suggests that ambient concentrations of this element may not be sufficiently elevated to adversely affect reproductive success and survival in selenium-tolerant poeciliids and pupfish.