A phenotypically interesting strain of cattle existed on the small island of Agersoe, on the west coast of Zealand, Denmark, in the beginning of the last decade. The cattle share a great resemblance to the extinct Danish breed, the Island cattle. The objective of this study was to genetically characterize the Agersoe cattle, using microsatellites, amplified fragment length polymorphism (AFLP) and mtDNA markers, and analyse the genetic variability within the breed and the genetic relationship to 14 European breeds with focus on the Red Danish and Jutland breed. The results show diversity in nuclear markers comparable to that of modern breeds and that the Agersoe cattle are separable from the two native breeds. The absence of inbreeding and the degree of genetic diversity are taken as a sign of recent admixture. The Agersoe cattle did not exhibit a consistent association with any of the European breeds. Several arguments based on this survey have been put forward in favour of characterizing the Agersoe cattle as being the last remnants of the Danish Island Cattle.