Otoliths, calcified inner ear structures, were collected from creek chubs (Semotilus atromaculatus) and green sunfish (Lepomis cyanellus) living in mountaintop mining-impacted and reference streams and analyzed for selenium (Se) content using laser ablation-inductively coupled mass spectrometry. Significant differences in otolith Se were found between the 2 fish species. Results from the present study suggest that a retrospective reconstruction of Se concentrations in muscle can be derived from Se concentrations in otoliths in creek chub but not green sunfish, exemplifying the importance of species differences when determining partitioning of Se among specific tissues. Green sunfish otoliths from all sites contained background (<1 μg/g) or low (1-4 μg/g) average concentrations of whole-otolith Se. In contrast, creek chub otoliths from the historically mined site contained much higher (≥5 μg/g) concentrations of Se than for the same species in the unmined site or for the green sunfish. These data suggest that body burdens of Se in fish can vary considerably over time and that both the timing of sampling and species choice could heavily influence Se assessments.