Conservationists are in need of objective metrics for prioritizing the management of habitats. For individual species, the threat of extinction is often used to prioritize what species are in need of conservation action. Using long-term monitoring data, we applied a Bayesian diffusion approximation to estimate quasi-extinction risk for 54 native fish species within six commercial navigation reaches along a 1350-km gradient of the upper Mississippi River system. We found a strong negative linear relationship between quasi-extinction risk and distance upstream. For some species, quasi-extinction estimates ranged from nearly zero in some reaches to one in others, suggesting substantial variability in threats facing individual river reaches. We found no evidence that species traits affected quasi-extinction risk across the entire system. Our results indicate that fishes within the upper Mississippi River system face localized threats that vary across river impact gradients. This suggests that conservation actions should be focused on local habitat scales but should also consider the additive effects on downstream conditions. We also emphasize the need for identification of proximate mechanisms behind observed and predicted population declines, as conservation actions will require mitigation of such mechanisms. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.