The lake minnow (Eupallasella percnurus) is critically endangered. In this paper we characterize the genetic properties of this fish over its range of occurrence in Poland and propose the use of this knowledge in its active protection. Twelve populations of lake minnow from across its range in Poland were investigated. 13 microsatellite loci were investigated to evaluate genetic variation and distance among populations. The magnitude of the genetic bottleneck or founder effects was investigated. In the studied populations, the allelic diversity and heterozygosity showed that genetic variation in this species is low. At most loci, only 2–3 alleles per population were detected. The average number of alleles detected across all loci was 35, and ranged from 24 to 53. The average observed heterozygosity (Ho) across all investigated loci was 0.38 (range 0.21–0.59); the average expected heterozygosity (He) was 0.36 (range 0.18–0.55). The populations remained in Hardy-Weinberg equilibrium. The average Garza-Williamson M index value for all populations was low (0.47), suggesting a reduction in genetic variation due to a founder effect or a genetic bottleneck. Genetic distance among populations was high or very high (FST range: 0.20–0.64; δμ2 range: 1.32–16.98); this was likely a consequence of low gene flow among isolated populations, a founder effect or other genetic bottleneck, and strong genetic drift. The large genetic differences among the investigated lake minnow populations are likely to also exist among other populations of this species, and knowledge of these differences should inform active protection programs based on translocation of wild or cultivated fish of this species. The method presented here can potentially be applied to any population of lake minnows or closely related species.