European bat lyssavirus 1 (EBLV-1) is widespread and frequent in European bats, particularly of the genus Eptesicus. However, other species can be seropositive, suggesting a complex ecology still mostly unknown. In Italy, EBLV-1 antibodies are described since 2012 in South Tyrolean Myotis myotis. This study provide phylogenetic evidence for either the current or the recent movement of M. myotis from continental Europe to South Tyrol across the Alpine valleys, which might allow for the introduction of LYSVs. Serological analyses confirmed antibodies against EBLV-1 in this bat across the entire region, showing marked seasonal pattern and a sharp peak of positivity in late summer. No statistical difference was detected between the maternity colonies investigated in either the likelihood for infection or the antibody titres. Indeed, South Tyrolean populations of M. myotis showed no significant genetic differentiation using nuclear and mitochondrial markers, supporting the existence of a regional meta-population and a low philopatric behaviour. This structure, never described for M. myotis elsewhere, well explains the spread and maintenance of LYSVs trough the movement of female bats between colonies. Since no virus was detected in the study, we cannot exclude that our serological data don’t derive from a cross-reaction with an unknown LYSV. However, because all LYSVs can cause clinical rabies in humans, this study has a strong impact on public health regardless of the viral species actually circulating. In addition, the unravelling of the peculiar genetic structure of South Tyrolean M. myotis is crucial to inform conservation strategies for this endangered species.