The genetic variability of Pantesco and other two Sicilian autochthonous donkey breeds (Ragusano and Grigio Siciliano) was assessed using a set of 14 microsatellites. The main goals were to describe the current differentiation among the breeds and to provide genetic information useful to safeguard the Pantesco breed as well as to manage Ragusano and Grigio Siciliano. In the whole sample, that included 108 donkeys representative of the three populations, a total of 85 alleles were detected. The mean number of alleles was lower in Pantesco (3.7), than in Grigio Siciliano and Ragusano (4.4 and 5.9, resp.). The three breeds showed a quite low level of gene diversity (He) ranging from 0.471 in Pantesco to 0.589 in Grigio. The overall genetic differentiation index (Fst) was quite high; more than 10% of the diversity was found among breeds. Reynolds' (D(R)) genetic distances, correspondence, and population structure analysis reproduced the same picture, revealing that, (a) Pantesco breed is the most differentiated in the context of the Sicilian indigenous breeds, (b) within Ragusano breed, two well-defined subgroups were observed. This information is worth of further investigation in order to provide suitable data for conservation strategies.