The two old world flycatcher species, the Pied and the Collared Flycatcher are ecologically and behaviourally similar except for their song. The species have a predominantly allopatric distribution but hybridize in areas of geographic overlap. The genetic differentiation between the two species detected by protein electrophoresis was low (Nei’s genetic distance 0.0006), and no species-specific alleles were found. Repetitive sequences in nuclear DNA investigated by restriction enzymes were also similar. A comparison of restriction fragment differences in mitochondrial DNA, however, showed a sequence divergence of 10% between the species. The karyotypes of the two species are similar, both possessing 80 chromosomes. Studies of hatching success in combination with DNA-fingerprinting studies of parentage showed that female interspecific hybrids are sterile whereas hybrid males are fertile. Asymmetrical hybrid fertility allows nuclear but prevents mitochondrial gene flow between the species. These results demonstrate the advantage of using a multi-methodological approach for analyzing genetic divergence and speciation.