We assessed the suitability of two nonnative poeciliid fishes—western mosquitofish (Gambusia affinis) and sailfin mollies (Poecilia latipinna)—for monitoring selenium exposure in desert pupfish (Cyprinodon macularius). Our investigation was prompted by a need to avoid lethal take of an endangered species (pupfish) when sampling fish for chemical analysis. Total selenium (SeTot) concentrations in both poeciliids were highly correlated with SeTot concentrations in pupfish. However, mean SeTot concentrations varied among fish species, with higher concentrations measured in mosquitofish than in mollies and pupfish from one of three sampled agricultural drains. Moreover, regression equations describing the relationship of selenomethionine to SeTot differed between mosquitofish and pupfish, but not between mollies and pupfish. Because selenium accumulates in animals primarily through dietary exposure, we examined fish trophic relationships by measuring stable isotopes (δ¹³C and δ¹⁵N) and gut contents. According to δ¹³C measurements, the trophic pathway leading to mosquitofish was more carbon-depleted than trophic pathways leading to mollies and pupfish, suggesting that energy flow to mosquitofish originated from allochthonous sources (terrestrial vegetation, emergent macrophytes, or both), whereas energy flow to mollies and pupfish originated from autochthonous sources (filamentous algae, submerged macrophytes, or both). The δ¹⁵N measurements indicated that mosquitofish and mollies occupied similar trophic levels, whereas pupfish occupied a slightly higher trophic level. Analysis of gut contents showed that mosquitofish consumed mostly winged insects (an indication of terrestrial taxa), whereas mollies and pupfish consumed mostly organic detritus. Judging from our results, only mollies (not mosquitofish) are suitable for monitoring selenium exposure in pupfish.