Smallmouth bass in the Susquehanna River Basin, Chesapeake Bay Watershed, USA, have been exhibiting clinical signs of disease and reproductive endocrine disruption (e.g., intersex, male plasma vitellogenin) for over fifteen years. Previous histological and targeted chemical analyses have identified infectious agents and pollutants in fish tissues including organic contaminants, mercury, and perfluorinated compounds, but a common causative link for the observed signs of disease across this widespread area has not been determined. This study examines 146 young-of-year smallmouth bass collected from 14 sampling sites in the Susquehanna River Basin, Pennsylvania, USA with varying levels of disease prevalence. Whole fish were extracted by a recently developed modification to the quick, easy, cheap, effective, rugged, and safe extraction method and analyzed by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. A targeted analysis was conducted to identify the presence and quantity of 127 known contaminants, including polychlorinated biphenyls, brominated diphenyl ethers, organochlorinated pesticides, and pharmaceutical and personal care products. A non-targeted analysis was conducted on the same data set to identify analytes of interest not included on routine target compound lists. Chromatographic alignment through Statistical Compare (ChromaTOF GC) was followed by Fisher ratio and principal component analysis to reduce the data set from thousands of peaks per sample to a final data set of 65 analytes of interest. Comparisons of these 65 compounds between Normal (no observed health anomalies) and Lesioned (observed health anomaly at time of collection) fish revealed increased levels of three chemical families in Lesioned fish including esters, ketones, and nitrogen containing compounds.