Myotis bucharensis is one of the least studied Palaearctic bat species, known from only three localities in Tajikistan and Uzbekistan and not reported since 1965. In autumn 2019, a male Myotis captured in Zerafshan river basin in Tajikistan was later identified as M. bucharensis based on tail and tibia proportions and strongly displacement of posterior small premolars. The identification was then confirmed by morphometric analyses supporting that M. bucharensis is specifically different but represents a part of the Myotis frater complex. Analyses of one mitochondrial (cyt b) and one nuclear gene (RAG2) were performed for the first time for M. bucharensis. According to these genetic results, this form is indeed a member of the «daubentonii» clade, which includes all known frater-like Myotis, and most likely represents a sister species to M. longicaudatus. Record of the alive specimen M. bucharensis has valuable implication for bat conservation in Tajikistan.