Indigenous cattle breeds represent a unique genetic resource, and understanding their variability, population structure and breeding units is important for their sustainable conservation. The endangered Jutland breed was widespread in Denmark in the eighteenth century, but decreased in population size following the introduction of modern farming. We investigated the impact of recent anthropogenic fragmentation of the breed by analysing 737-bp mitochondrial DNA and 23 microsatellites in 207 individuals. The results revealed the Jutland breed as a unique genetic entity with high levels of genetic diversity, and only limited introgression from other black-pied breeds. The data reflected the impacts of fragmentation and restricted gene flow in breeds with small segregated herds, and revealed the rapid differentiation of herds resulting from genetic drift. The application of a management strategy that conserves diversity and minimizes increase in inbreeding is important for the future conservation of the Jutland breed and other indigenous cattle breeds.