This paper presents the results of an analysis of about four hundred skulls of Myotis mystacinus and M. brandtii of the Holocene age from the Záskočie Cave (Central Slovakia). A review of criteria for species determination is presented, including a new criterion concerning the morphology of P4 roots, as well as the results of a statistical survey of skull dimensions and correlation and regression analyses. The author concludes that the above mentioned bats are not sibling species, but that they belong to different subgenera of the genus Myotis.