ABSTRACT Recent morphological and molecular studies suggested the existence of several undescribed species within the genus Barbatula . The stone loach ( Barbatula barbatula ) is considered, according to the Italian Red List, as native in Northern Italy and classified as vulnerable (VU), having a limited and fragmented distribution from Lombardy to Friuli‐Venezia Giulia regions. In the present study, 248 specimens of Barbatula sp., collected from 17 sampling sites in Italy—spanning its entire known distribution area—and from one site in Austria, were analysed by sequencing the Cytochrome C Oxidase I (COI) and the Cytochrome B (CytB) mitochondrial regions. Sequencing results were then compared with reference samples from the literature. Three highly divergent mitochondrial lineages were observed in Italian populations, which can be associated with three different species: Barbatula pironae in Friuli‐Venezia Giulia, Barbatula fluvicola in Trentino‐Alto Adige and Lombardy, and Barbatula aff. barbatula coexisting with the latter in Lombardy. The three species, with the first having a distribution limited to the upper Adriatic area, and the other two having a wider distribution north of the Alps, should therefore be considered as different Management Units. Therefore, the integration of the Italian freshwater fish species checklist and the update of their taxonomy are strongly advised. Our data together with other available evidence suggest that the three species are likely native to Italy, and hence a revision or definition of their conservation status might be needed.