The aims of this study were to assess the genetic variability in the exon 1 of the κ-casein gene in four Italian horse populations (Italian Saddle horse, Italian Trotter, Italian Heavy Draught horse, and Murgese horse) and in a sample of Martina Franca donkey by estimating genotype, allele and haplotype frequencies, as well as several population genetic indices. Genotyping of the selected polymorphisms was performed using the PCR-RFLP technique with two restriction enzymes: PstI and BseYI aimed to discover the presence of c.-66A>G and c.-36C>A polymorphism, respectively. Both these loci were found to be polymorphic in horses with some differences depending on the breed. No genetic variability was observed in Martina Franca donkey breed. In the equine species no selective pressure for milk purpose was performed, therefore the polymorphisms at milk protein loci were mainly considered as result of natural selection or as indirect consequence of selection oriented to increase body size or to improve conformation.