Recently described Myotis alcathoe has been recorded in Ukraine for the first time and at eight new localities in three distant and geographically diverse areas of Poland (the mountains, the uplands and the lowlands). This data extends its distribution range in Central Europe and confirms it as a native breeding species in Poland. Specific identification of bats was confirmed by sequencing ND1 gene of mtDNA. In Poland its reproduction has been recorded in Łężczok reserve, Silesian Foothill and in Roztocze National Park. Bats were observed mostly within old broadleaved and mixed forests, near water, at altitude ranging from 112 to 736 m a.s.l. The only known roosting site for the species is a cave which is used both in spring and during the swarming period. In Poland the species co-occurred with both M. brandtii and M. mystacinus or with M. brandtii at all sites.