We report the results of the elemental and stable isotopic analyses, as well as the composition of stomach contents, of Arctic grayling (Thymallus arcticus), an ecologically important resident freshwater sport and subsistence fish in the Fortymile River Mining District of the Interior Highlands Ecoregion in eastern Alaska. These data are presented here as a data compilation with minimal interpretation or discussion. Further analyses of the data will be presented elsewhere. The study area has been mined for placer gold for over a century and is currently experiencing renewed mineral exploration activity. The results for the analysis of 40 inorganic elements are reported for grayling muscle (fillet) tissue, liver tissue, and stomach contents from 34 individuals caught at 11 sites within the watershed. The 11 sites were classified as occurring within the following lithologies: metavolcanic (7 sites), metasedimentary (3 sites), and granitic intrusion (1 site). This information (along with fish tissue stable isotope data) is critical in the assessment of the influence of regional lithology on the fish chemical composition, especially the trace metal content. We report the nitrogen, carbon, and sulfur stable isotope composition of muscle samples. Nitrogen isotopes appear homogeneous (d15N = 7.6 to 9.7 permil) whereas carbon and sulfur isotope compositions of the same samples span a range from d 13C = ?33.1 to ?25.8 permil, and d 34S = ?8.4 to 8.2 permil. Stomach content material was examined for the occurrence and frequency of macroinvertebrate composition and diversity in three individual fish. Results showed a high degree of diversity with 9 to 15 invertebrate taxa; both aquatic and terrestrial forms were represented.