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Fountain Creek in Colorado USA is a major tributary that confluences with the Arkansas River at Pueblo, Colorado, the result being the tributary's influence on Arkansas River water quality affecting down-stream users. In a previous study, we found that bryophytes (aquatic plants) accumulated selenium in Fountain Creek watershed and this finding prompted us to investigate the extent of the metalloid in the whole-body tissues of fish. One hundred 11 fish (six species) were collected and analyzed for Se by inductively-coupled plasma emission mass spectrometry. Analysis of all analytical data also showed mercury in all of the fish whole bodies and selected tissues. There was a general increase in selenium but a decrease in mercury in fish with downstream travel-distance. The highest whole-body selenium was in Pueblo, Colorado (3393 µg/kg, dry weight; 906 µg/kg, wet weight); the highest mercury in fish was in the Monument Creek tributary north of Colorado Springs, Colorado (71 µg/kg, dry weight; 19 µg/kg, wet weight). In four tissues of 11 female fish captured, selenium was highest in the livers at eight sites but highest in the ovaries at three sites. Mercury was highest in the epaxial muscle at all sites. Selenium availability could be due to the watershed lithology and land uses; however, mercury could be carried by atmospheric deposition from coal-fired power plants and historic mining activities. Selenium in fish tissues and water samples were compared to U.S. national water quality criteria.