show Abstracthide AbstractThe field vole is a complex of three cryptic species. The short-tailed field vole (Microtus agrestis) is present over much of Eurasia, the Mediterranean field vole (Microtus lavernedii) is found in southern Europe, and the Portuguese field vole (Microtus rozianus) is limited to western Spain and Portugal. Previous studies showed discordance of mitochondrial and nuclear gene trees and suggested that the Portuguese field voles diverged from Mediterranean and short-tailed field voles ca. 70 thousand years (ka) ago, while the two latter separated about 25 ka ago during the Last Glacial Maximum (LGM). It was suggested that isolation in the different refugial areas was the main factor causing the genetic differentiation of these populations. To elucidate evolutionary history of the field vole species complex, we generated mitochondrial genomes from 75 ancient and 46 modern specimens and nuclear genomes from 10 ancient voles, dated up to ca. 70 ka, and several modern specimens. The calibrated mitochondrial phylogeny shows that the northern lineage diverged from the Portuguese and Mediterranean lineages ca. 200 ka ago, while the latter diverged about 100 ka ago. Preliminary analyses of nuclear genomes also suggest that Mediterranean and short-tailed field voles diverged much earlier than previously estimated, probably before the Last Glaciation (115–11.7 ka ago), but they shared a similar evolutionary history with severe bottlenecks during the LGM. Further analyses will search for the primary factors that contribute to the divergence of the field vole species complex.