Scolitantides orion is a butterfly species threatened in many European countries. In Poland, it survived in a single highly isolated area (Vistula River valley), which is an example of the dramatic decline in the population number. We studied the two largest remaining populations inhabiting opposite banks of the river. Mark-release-recapture studies showed that both populations were small, and they fluctuated in numbers, but adult individuals were twice as numerous on the western site. Genetic analyses were carried out using a mitochondrial (COI, ND5) and nuclear markers (Wgl, EF-1α, and microsatellite loci). We found out that genetic variation was low at both sites but higher in the smaller eastern population. This pattern is likely to be better explained by past distribution, when the butterfly, as a continental species used to be much more widespread in the east. However, the genetic differentiation between populations was low. This could suggest that the existing gene flow is facilitated by dominant regional wind direction, which may also contribute to a better genetic condition of the western population. Finally, a comparison of the obtained COI sequences with others available enabled us to reveal the phylogeographic pattern of the S. orion from different localities within its range.