The serotine bat, Eptesicus serotinus is the most frequently rabies-infected (European bat lyssavirus 1-type, EBLV-1) bat species in Europe. To confirm Lyssavirus infection of this bat in Poland, we tested for the presence of rabies virus RNA from oropharyngeal swabs using RT-PCR. There was a 0.9% (two out of 212 individuals) level of infection within the overall population of serotine bats studied. However, an appreciation of the potential for pathogen transmission and disease risk requires an understanding of the dispersal of the primary host, and any large-scale geographic barriers that may impede gene flow. Thus, we also studied the patterns of bat dispersal via population genetics using nuclear (seven microsatellite loci) and mitochondrial (mtDNA control region) markers, examined in 12 subpopulations distributed across the country. Molecular analyses of microsatellite loci indicated high genetic diversity at all sites (heterozygosity observed, H_o = 0.53–0.78), and extremely weak genetic structure in the Polish population of the species. The overall F_ST was 0.012 (95% confidence interval: 0.006–0.020), and pairwise values ranged from 0.00 to 0.05. Only 22% of individuals were assigned to the subpopulation from which they were sampled. The Bayesian approach implemented in STRUCTURE also confirmed that all examined subpopulations should be treated as a single group, indicating a high level of gene flow. There was some evidence for female philopatry (genetic differentiation was greater in maternally-inherited mtDNA than nuclear DNA) and male-biased dispersal, e.g., H_o and the variance of mean assignment were significantly higher in males than in females. Twelve individuals (seven females and five males) were identified as potential first generation migrants. Their migration routes ranged from 60–283 km in females ( ± SE = 177.9 ± 29.37) to 27–385 km in males (206.4 ± 58.95); surprisingly, no sexual differences were observed and this finding suggests that female-mediated gene flow may occur. MtDNA also produced a strong genetic signal for the demographic expansion (Fu's F_S statistics, F_S = -26.30, P < 0.01 and a star-shaped haplotype network), which took place roughly 33,000 years BP, i.e., before the Last Glacial Maximum. The genetic uniformity of the Polish population implies that there is no migration barrier to EBLV-1, at least within the country, and the potential threat of rabies virus spreading via migration of infected animals may be higher than previously thought.