AbstractIn the developed countries donkey has lost its main function as draft animal because of the mechanization in agri-culture; as a consequence donkey population was greatly reduced. According to SAVE monitoring institute, three of the eight Italian endangered donkey breeds are native of Sicily (Ragusano, Pantesco, Grigio Siciliano). Urgent safeguard plans are required. The aim of this work is to investigate the distribution of genetic diversity and the relationships among the three Sicilian autochthonous donkey breeds using a set of microsatellite markers. A total of 116 blood samples (61 Ragusano, 39 Pantesco, 16 Grigio Siciliano) were collected in 9 herds all over Sicily. Representative samples of Ragusano and Grigio donkey populations consist of unrelated individuals, whereas the sample of Pantesco represents nearly the entire studbook-registered population managed by "Ispettorato Ripartimentale delle Foreste di Erice (TP)" in the "Azienda S. Matteo". Genomic DNA was amplified at 11 microsatellite..