A major contaminant of concern for mountaintop removal/valley fill (MTR/VF) coal mining is selenium (Se), an essential micronutrient that can be toxic to fish. Creek chubs (Semotilus atromaculatus), green sunfish (Lepomis cyanellus), and composite insect samples were collected in March-July, 2011–2013 at two sites within the Mud River, West Virginia. One site (MR7) receives MTR/VF coal mining effluent, while the reference site (LFMR) does not. MR7 water had significantly higher concentrations of soluble Se (p < 0.01) and conductivity (p < 0.005) compared to LFMR. MR7 whole insects contained significantly higher concentrations of Se compared to LFMR insects (p < 0.001). MR7 creek chubs had significantly higher Se in fillets, liver, and ovary tissues compared to LFMR samples (p < 0.0001, p < 0.0001, and p < 0.02, respectively). MR7 green sunfish fillets contained significantly higher Se (p < 0.0001). Histological examination showed LFMR creek chub gills contained a typical amount of parasitic infestations; however MR7 gills contained minimal to no visible parasites. X-ray absorption spectroscopic analyses revealed that MR7 whole insects and creek chub tissues primarily contained organic Se and selenite. These two species of Mud River fish were shown to specifically accumulate Se differently in tissues compartments. Tissue-specific concentrations of Se may be useful in determining potential reproductive consequences of Se exposure in wild fish populations.