We investigated potential adverse impacts of urban-derived environmental contaminants, such as pharmaceuticals, steroids, surfactants and plasticizers, on Longnose Dace (Rhinichthys cataractae) along two rivers, the Bow and Elbow Rivers, in the City of Calgary. Fish were sampled to evaluate physiological and morphological endpoints associated with reproduction and development, including adult sex ratios, changes in body and organ weight, and gonad malformation. Significant male bias was observed downstream of three wastewater treatment plants (WWTPs) on the Bow River, and significant female bias was observed on the Elbow River, suggesting the presence of environmental contaminants with hormone-like activity, dependent on location. To investigate the mechanisms of adverse fish health effects we quantified the expression of liver vitellogenin, estrogen receptor alpha, cytochrome P450, and insulin-like growth factor-1. Decreased IGF1 and ERα expression levels were observed downstream of WWTP effluent in the Bow River, while increased vitellogenin and ERα expression levels were noted in the Elbow River within Calgary. Results support the hypothesis that waterborne environmental contaminants may be responsible for the adverse health effects, such as biased sex ratios, of Longnose Dace within the City of Calgary.