Species reintroduction efforts can improve the recovery of imperiled species, but successful implementation of this conservation strategy requires a thorough understanding of the abiotic and biotic factors influencing species viability. Species interactions are especially understudied, in particular by omitting the effect of imperfect detection on negative, neutral, or positive associations within a community. Using repeat surveys from 5 southern Ontario, Canada, Great Lakes tributaries, we quantified species co‐occurrence patterns with the eastern sand darter (ESD) (Ammocrypta pellucida), listed as federally threatened, and characterized how imperfect detection during sampling can influence inference regarding these relationships. We used a probabilistic framework that included 3 approaches of increasing complexity: probabilistic co‐occurrence analysis ignoring imperfect detection; single‐species occupancy models with subsequent co‐occurrence analysis; and 2‐species occupancy models. We then used our occupancy models to predict suitable sites for potential future reintroduction efforts while considering the influence of negative species interactions. Based on the observed data, ESD showed several positive associations with co‐occurring species; however, species associations differed when imperfect detection was considered. Specifically, a negative association between ESD and rosyface shiner (Notropis rubellus) was observed only after accounting for imperfect detection in the Grand River. Alternatively, positive associations in the Grand River between ESD and northern hogsucker (Hypentelium nigricans) and silver shiner (Notropis photogenis) were observed regardless of whether imperfect detection was accounted for. Our models predicted several potential reintroduction sites for ESD in formerly occupied watersheds with high levels of certainty. Overall, our results demonstrate the importance of investigating imperfect detection and species co‐occurrence when planning reintroduction efforts.