Abstract. 1. The regal fritillary butterfly, Speyeria idalia Drury 1773, was once widespread across eastern North America, but has declined significantly and rapidly over the past half-century. Although more stable in the western portion of its range, only two populations survive east of the Great Lakes, one in eastern Pennsylvania and the other in Virginia.
2. Previous studies have found that the remnant Pennsylvania population is genetically differentiated from populations in the west, and have suggested the designation of separate eastern and western subspecies. However, the historical pattern of genetic variation from which the current distinctness of the Pennsylvania population has arisen was not known, nor was the relationship with the remnant Virginia population.
3. We amplified and sequenced two mitochondrial loci (COI/II and ND4) from preserved specimens to infer historical patterns of genetic variation in this species, and we used non-lethally obtained tissue samples to assess the relationship of the two eastern remnant populations.
4. We found very consistent patterns between the two loci. Both had a very shallow haplotype network with few mutations separating most haplotypes. At both loci, we observed distinct groups of haplotypes in the western and far eastern (i.e. New England) portions of the range; a region of transition was centred on Ohio, western Pennsylvania, and the Virginias, where both groups, and intermediate haplotypes, were represented.
5. Importantly, the extant Virginia population shared haplotypes with western populations of S. idalia and not with the extant Pennsylvania population. We discuss the implications of this result for the taxonomy and translocations/introductions of the species.