Reproductive and oxidative stress biomarkers have been recommended as tools to assess the health of aquatic organisms. Though validated in the laboratory, there are few studies that tie a change in gene expression to adverse reproductive or population outcomes in the field. This paper looked at 17 streams with varying degrees of urbanization to assess the use of biomarkers associated with reproduction or stress in predicting reproductive success of fathead minnows. In addition, the relationship between biomarkers and water quality measures in streams with varying degrees of urbanization was examined. Liver vitellogenin mRNA was correlated with reproduction within a period of 11 d prior to sampling irrespective of habitat, but its correlation with egg output declined at 12 d and beyond indicating its usefulness as a short-term biomarker but its limits as a biomarker of total reproductive output. Stress biomarkers such as glutathione S-transferase may be better correlated with factors affecting reproduction over a longer term. There was a significant correlation between GST mRNA and a variety of anthropogenic pollutants. There was also an inverse correlation between glutathione S-transferase and the amount of the watershed designated as wetland. Egg production over the 21-d was negatively correlated with the amount of urbanization and positively correlated to wetland habitats. This study supports the development of multiple biomarkers linking oxidative stress and other non-reproductive endpoints to changes in aquatic habitats will be useful for predicting the health of fish populations and identifying the environmental factors that may need mitigation for sustainable population management.