In English The Chiffchaff Phylloscopus collybita is represented in Sweden by two different subspecies; the northern well-established abietinus and the southern recently established collybita which has expanded its range northward during the past two decades. At present, an area approximately 500 km wide separates the two subspecies. In order to document differences between the northern and southern populations we compared morphology, vocalisation, habitat choice, and neutral genetic variation in mitochondrial (mt) DNA and at four microsatellite loci of 30 male Chiffchaffs from each subspecies. Our results show significant differences in several morphological traits and in song. Playback experiments revealed a significant difference in aggressive behaviour depending on which population-specific song that was played to the birds. Mitochondrial DNA was geographically structured with similar to 90% of the birds carrying a mt haplotype matching their sample population. No allelic differences at the microsatellite loci were found between populations. Our data demonstrate a substantial differentiation between the northern and southern populations despite gene flow, clearly separating them into the subspecies abietinus and collybita.