Phylogenetic relationships of European Maniola butterflies are reconstructed using molecular sequences from two regions of the mitochondrial DNA, cytochrome oxidase subunit I (COI) and cytochrome b (Cytb). A total of 988 base pairs (486 for Cytb, and 502 for COI) were aligned for 15 individuals of Maniola and an outgroup, species. The phylogenetic tree obtained through Bayesian inference analysis of the combined data sets shows evidence that the island endemic M. chia is indistinguishable from M jurtina on the basis of the mtDNA genes studied. Net nucleotide divergence between M jurtina and M chia is 0.4%, but 2% between the M jurtina and the M nurag clade. A phenetically distinct entity of individuals from Sardinia appears to be a hybrid between M nurag and M jurtina. The southern and northern European ecotypes of M jurtina, which differ in the summer aestivation period of the southern type, are not structured genetically at the level of coding mtDNA genes. Divergence time between M nurag and M jurtina was estimated to be 1.1 to 1.2 million years. Speciation most likely took place in the early Pleistocene as a consequence of the isolation of Sardinia, when the sea reflooded the Mediterranean basin after the Messinian crisis (about 5 million years ago).