Phengaris alcon is an endangered, ant-associated butterfly found, amongst other places, in Denmark, where it has undergone a severe decline during the last century. However, the population genetic consequences of this decline remain unknown. To explore past and current patterns in population structure in relation to the decline, we analyzed DNA microsatellite data from 184 recent and 272 historical P. alcon specimens from 44 spatiotemporal locations in Denmark. We thus generated the most temporally and spatially comprehensive population genetic dataset for P. alcon in Denmark so far. Our results for the Bayesian population assignment of recent samples revealed three major current genetic clusters: western Jutland, northern Jutland, and the island of Læsø. Estimates of genetic diversity showed signs of inbreeding in several extant populations. When including data from museum specimens, only a single locatSion showed a decline in heterozygosity between 1967 and 2021. We suggest that the two distinct clusters in western and northern Jutland indicate two temporally separated Holocene colonizations of Denmark, the latter of which may have been aided by changes in agricultural practice in the late Neolithic period. The unique genetic signature of the Læsø populations may be a result of the admixture of northern Jutland and western Swedish populations.