Variability at 20 microsatellite loci was examined to assess the population genetic structure, gene flow, and effective population size (Ne) in three populations of three-spined stickleback (Gasterosteus aculeatus) from the upper basin of the Miño River in Galicia, NW Spain, where this species is threatened. The three populations showed similar levels of genetic diversity. There is a significant genetic differentiation between the three populations, but also significant gene flow. Ne estimates based on linkage disequilibrium yielded values of 355 for the Miño River population and 241 and 311 for the Rato and Guisande Rivers, respectively, although we expect that these are overestimates. Ne estimates based on temporal methods, considering gene flow or not, for the tributaries yielded values of 30–56 and 47–56 for the Rato and Guisande Rivers, respectively. Estimated census size (Nc) for the Rato River was 880 individuals. This yielded a Ne/Nc estimate of 3–6 % for temporal estimation of Ne, which is within the empirical range observed in freshwater fishes. We suggest that the three populations analyzed have a sufficient level of genetic diversity with some genetic structure. Additionally, the absence of physical barriers suggests that conservation efforts and monitoring should focus in the whole basin as a unit.