Highly endangered local breeds are considered important not only for the maintenance of their genetic diversity for future survival but also because they regarded as part of the cultural heritage of the local and national communities. Using pedigree data and an analysis of 18 microsatellite loci we investigated the genetic diversity of a private (commercial) indigenous Skyros horse population, reared in an insular region of North-Western of Greece. The overall average animal inbreeding value reached 24%. Concerning average inbreeding value over non founding animals, it was estimated to 0.013, while the corresponding value over inbred animals were 0.13.The mean number of alleles per locus amounted to 3.72, ranging between 1 and 7 alleles. The average observed heterozygosity was 0.57. Taking into account the inbreeding estimated index, an average heterozygote deficit (Fis) of -0.09 was noted (P<0.05). Although the population maintained reasonable levels of genetic diversity, well studied inbreeding strategies should be implemented, in order to reduce the loss of genetic variability, to avoid extinction and further genetic drift of the population.