The Manchega sheep breed is an autochthonous dairy breed of Spain, which has two colour varieties, the white and the black. There are only a few herds of black animals, and as a result, this variety is considered in Spain to be a special protected endangered breed, receiving funds for its conservation. Genetic variability at 15 microsatellite loci and two mutations at MC1-R locus was analysed at three different hierarchic levels in the data of the Manchega Spanish sheep breed (1) the breed itself, i.e. the whole data set without division into colour varieties and flocks; (2) the two colour varieties; and (3) five flocks (three blacks and two whites). Allele frequencies and heterozygosity revealed high genetic variation in the black variety despite its small population size. Noticeably, the Black3 population showed the wild-type allele for the MC1-R locus related to white colour at a significant frequency of 0.208. The degree of population subdivision calculated between varieties and populations from FST indices was around 4% of the total variability. The allele frequencies estimated were used to assign an unknown genotype of an individual to the colour variety population or flock (using their genotype frequencies as a reference) with a high degree of accuracy (99% by the Bayesian method using six microsatellites in variety assignment). In this way, although Black3 animals are black in phenotype, this population is near to the white population in genotype, because only 16.6% were assigned to the black variety. Values of DReynolds (0.038) and Dm (0.092) genetic distances indicated a clear differentiation between the two varieties. DReynolds distance between flock pairs revealed that White1/White2 and Black1/Black2 populations are very close, while Black3 is closer to the white populations. In conclusion, Manchega sheep breed colour varieties show good diversity measures that can permit a higher selection pressure without increasing inbreeding related to the whole population (FIT = 0.045) and within population (FIS = 0.009). Furthermore, results of heterozygosities, individual assignment test, and distance values indicate a crossbreeding of Black3 and White animals. As a result, some of the Black3 animals are not suitable for use in the conservation-breeding program of the black variety. © 2005 Elsevier B.V. All rights reserved.