Summary This study describes the isolation and molecular characterization of Mammalian orthoreovirus (MRV) in microbats. Faecal samples and dead individuals available from rehabilitation centres or collected from known roost sites were virologically tested. In total, 112 carcasses of bats found dead, and 44 faecal samples were analysed. Nineteen viral strains were isolated by in vitro cell culture from faecal and tissue samples of different bat species (Pipistrellus khulii, Tadarida teniotis, Rhinolophus hipposideros and Vespertilio murinus), and they were morphologically identified as reoviruses by negative staining electron microscopy observation. The definitive assignment of all isolates to MRV was confirmed by RT-PCR assays targeting the L1 gene. Through a multiplex RT-PCR assay targeting the S1 gene, we typed 15 of 19 isolates as MRV type 3. Partial L1 (416 bp) and complete S1 (1416 bp) sequences of the isolates were analysed and compared with those of reference strains obtained from GenBank, belonging to the three serotypes. Molecular analysis of the S1 gene revealed that the amino acid residues associated with neurotropism (198-204NLAIRLP, 249I, 350D and 419E) were highly conserved among the Italian bat strains. These results suggest that potentially neurotropic MRV type 3 strains are widespread among Italian bats. Furthermore, the identification of MRV type 3 in bat species such as Pipistrellus Khulii, which is common in urban areas and known for its close contact with humans, underlines the need for vigilance.