The distribution of species and species diversity can be affected by vicariance or dispersal. To understand their role in shaping species distribution and population structure these two processes must be estimated within and among populations. I analysed large-scale variation in the call structure of the Ryukyu Scops Owl Otus elegans. This owl is distributed over a 1200-km range, and only inhabits islands. Within this range, I studied this species across 22 continental islands of the Ryukyu Archipelago and two oceanic islands. The study aimed to assess whether there is variation in the acoustic structure of Owl hoot calls within islands, among major groups of islands and across a large area comprising a major biogeographical barrier (the Kerama Gap). The acoustic structure of calls was homogeneous within islands and among major island-groups. Acoustic differentiation, however, increased over longer geographical distances of up to about 1200 km. The acoustic structure of hoots of the Ryukyu Scops Owl populations was clearly divided into two groups, north and south of the Kerama Gap. It is suggested that the Kerama Gap acted as a biogeographical barrier and contributed to the differentiation between the two major island-groups. It is likely that this difference developed during the fragmentation of a widespread ancestral population by vicariant isolating events. There was also evidence of an effect of dispersal on vocal differentiation in subspecies inhabiting the two oceanic islands.