In a survey of bats from La Rioja (Spain), several specimens of the mystacinus group were captured at different mountain localities. Genetic and morphologic analyses have revealed the presence of two lineages within this group in La Rioja. The lineages have been identified as corresponding to two different species: Myotis mystacinus sensu stricto and the recently described M. alcathoe. Both species were found using the same nocturnal refugia (caves) and the same forest habitats. This study extends the distribution of M. alcathoe west and southwards and adds a new mammal species to the Iberian fauna.