There are two karyotypes among Japanese populations of the leaf beetle Chrysolina aurichalcea. In “Type 31” beetles, the diploid number of chromosomes is 2n=32 in females and 31 in males, while in “Type 41” individuals, the diploid number of chromosomes is 2n=42 in females and 41 in males. Both karyotypes occur allopatrically or parapatrically in the wild, depending on location; generally, Type 41 beetles inhabit mountainous areas and Type 31 beetles occur on lowland plains. To assess genetic differentiation among C. aurichalcea populations and between the two karyotypes, we collected 606 individuals throughout Japan, determined the karyotypes of 203 individuals, and examined sequence variation in the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene for all 606 individuals. Karyotypic analysis indicated that hybrids between the two karyotypes are rare. A haplotype phylogeny shows that neither karyotype is monophyletic. These results may be explained by ancestral polymorphism and/or interkaryotype hybridization. Population genetic analysis showed that genetic divergence between the two karyotypes is relatively low, and similar to variation within karyotypes. Our data suggest that chromosomal changes occurred rapidly and recently. We also discuss the phylogeography of this species and the association between speciation and chromosomal rearrangements.