The marsh fritillary (Euphydryas aurinia) is a critically endangered butterfly species in Denmark known to be particularly vulnerable to habitat fragmentation due to its poor dispersal capacity. We identified and genotyped 318 novel SNP loci across 273 individuals obtained from 10 small and fragmented populations in Denmark using a genotyping-by-sequencing (GBS) approach to investigate its population genetic structure. Our results showed clear genetic substructuring and highly significant population differentiation based on genetic divergence (F_ST) among the 10 populations. The populations clustered in three overall clusters and due to further substructuring among these, it was possible to clearly distinguish six clusters in total. We found highly significant deviations from Hardy-Weinberg equilibrium due to heterozygote deficiency within every population investigated which indicates substructuring and/or inbreeding (due to mating among closely related individuals). The stringent filtering procedure that we have applied to our genotype quality could have overestimated the heterozygote deficiency and the degree of substructuring of our clusters but is allowing relative comparisons of the genetic parameters among clusters. Genetic divergence increased significantly with geographic distance, suggesting limited gene flow at spatial scales comparable to the dispersal distance of individual butterflies and strong isolation by distance. Altogether, our results clearly indicate that the marsh fritillary populations are genetically isolated. Further, our results highlight that the relevant spatial scale for conservation of rare, low mobile species may be smaller than previously anticipated.