Man-made structures, and habitat changes they impose, affect how fish are able to move up and downstream, between the channel and floodplain, and from habitats with unfavorable abiotic conditions to those that promote survival and reproductive output. Here we show that unidirectional stream-flow and dams affect patterns of effective population size (Ne) and genetic diversity in Rio Grande silvery minnow; a species with buoyant eggs that drift with river flow. We used archived DNA from 25 annual collections and targeted amplicon sequencing to test several predictions including that Ne and genetic diversity would be reduced upstream and increase in downstream reaches, and that augmentation and upstream stocking would disrupt these patterns. We found that Ne is reduced in the upstream-most reach and that there is a strong correlation between Ne in this reach and range-wide Ne suggesting that processes that act to reduce Ne and genetic diversity upstream, have a disproportionate impact on the population as a whole. In the absence of population augmentation, allelic diversity was reduced upstream and stocking with captive reared fishes in this reach restored diversity while other reach-specific patterns persisted despite augmentation. Up- to downstream trends in diversity and Ne imply that there is no longer sufficient spawning and retention in the natal reach to maintain large Ne and diversity is eroded through genetic drift. Movement of juveniles and adults and ongoing stocking may be insufficient to replenish depleted populations, maintain large Ne and genetic diversity. These results underscore a need for fish passage and habitat restoration promoting egg/larval retention.