Environmental compounds with estrogen- or antiestrogen-like activity can enter rivers from multiple sources, including municipal wastewater and agricultural runoff. We used longnose dace (Rhinichthys cataractae) to investigate exposure to compounds with estrogen-like activity, which we measured in water at multiple sites in the Oldman and Bow rivers (AB, Canada). We evaluated changes in vitellogenin mRNA with quantitative reverse transcription-polymerase chain reaction, then compared vitellogenin levels to sex ratios and fish performance indices to assess how exposure to compounds with estrogen-like activity affects longnose dace populations. Vitellogenin levels were elevated at least 59 to 110 km downstream of municipalities. In the Oldman River, increased vitellogenin expression and female-biased sex ratios suggest severe endocrine disruption, likely resulting from the combined impacts of municipal wastewater, agriculture, and large cattle operations within the basin. In the Bow River, municipal wastewater may be the major source of compounds with estrogen-like activity that affect longnose dace. The sex ratios were not heavily skewed, as in the more agriculturally influenced Oldman River. We detected organic contaminants in river samples at every site, but the highest concentrations were found downstream of municipalities and in areas with intense agriculture. Vitellogenin levels and sex ratios of longnose dace suggest basin-wide exposure to compounds with estrogen-like activity. Our results demonstrate that it is important to assess rivers at large spatial scales to detect fully the impacts of municipal wastewater and agriculture on fish populations.