Genetic variability and population structure in osprey were studied using DNA microsatellite markers. Special emphasis was placed on the subspecies living in the Afro-Palearctic (Pandion haliaetus haliaetus). For comparative purposes, American osprey subspecies (P. h. carolinensis, P. h. ridgway) and Indo/Australian subspecies (P. h. cristatus) were included in this analysis. Twenty DNA microsatellite loci were analysed across a total of 200 individuals. Cluster analysis of genetic distances generally grouped populations of osprey in accordance with their subspecific designation and with previous results from mtDNA analysis. Ospreys from America and Australia were clearly separated from P. h. haliaetus suggesting a more ancient isolation which prevented recent gene flow across these groups. Within P. h. haliaetus, significant genetic differentiation was found between populations in northern and southern Europe, suggesting that the Afro-Palearctic group is structured into two interconnected entities (Mediterranean and continental Europe). Population structuring was supported by an assignment test and by analysis of allele-sharing among individuals. At the Mediterranean scale, no significant differences of allelic information were found between populations. Behaviours such as dispersal, migration and philopatry seem to have played simultaneously and in contrary directions in shaping the genetic structure and diversity of populations. Our results provide essential information for reconstructing gene flow and genetic variability among osprey populations at different scales, which call for caution in the proactive management and conservation of the species, namely in the Mediterranean area.