The Total Maximum Daily Load (TMDL) for selenium in the freshwater drainages to Newport Bay, California, is being developed based on selenium concentration in the tissues of fish and bird eggs. This paper demonstrates the use of fish mesocosms and monitoring results to facilitate the comparisons of selenium contamination across fish species and areas of the watershed with differing fish assemblages. In this watershed, mosquitofish dominate across all the small, upper watershed drainages while sunfish family species dominate in deeper, ponded reaches of the lower watershed. Mesocosms were used to hold mosquitofish in ponds where they did not occur to compare their concentrations of bioaccumulated selenium to the tissue selenium of resident bluegill sunfish of the same pond. The caged fish were allowed to bioaccumulate selenium over time to achieve tissue concentrations at equilibrium conditions to compare as a ratio to resident bluegill. Those results were compared to the ratio of tissue concentrations from a later sampling of the same pond when the two species were found to co-occur for the first time. The ratios were brought into agreement only after altering assumptions of time to achieve equilibrium in bioaccumulated selenium for the transplanted mosquitofish and extrapolation of the mesocosm results. The technique demonstrates important considerations for the use of mesocosms to facilitate comparisons between allopatric species in terms of selenium bioaccumulation. A careful consideration of trophic level of the caged fish was found to be critical in setting the total time of bioaccumulation as part of the experimental design needed to achieve equilibrium tissue concentrations.