Boloria eunomia is a boreo-montane butterfly species suffering from habitat loss and isolation in the relictual part of its distribution range. Small populations persist in habitats scattered on plateaux or low mountains in western, central and southern Europe. Quantifying gene flow within and between these remnant populations is thus a crucial point to properly delineate metapopulations, to understand their dynamics and hence to design appropriate conservation plans for this butterfly species. We developed primers for the amplification of 15 microsatellites loci for B. eunomia. Thirteen loci were grouped in 2 multiplexes and amplified in 50 individuals from 5 populations to validate their use in population genetics. Gene diversity was on average 0.63 across populations. Null alleles and recurrent Hardy–Weinberg disequilibrium were detected in 7 loci. However, Fst estimates after correction for the presence of null alleles were highly correlated (0.91) to Fst estimates without correction. The loci developed here are thus usable for fine scale population genetic analyses and represent a very useful tool to quantify gene flow within and between metapopulations of B. eunomia.