Abstract. This study aimed at screening genetic diversity and differentiation in four horse breeds raised in Tunisia, the Barb, Arab-Barb, Arabian, and English Thoroughbred breeds. A total of 200 blood samples (50 for each breed) were collected from the jugular veins of animals, and genomic DNA was extracted. The analysis of the genetic structure was carried out using a panel of 16 microsatellite loci. Results showed that all studied microsatellite markers were highly polymorphic in all breeds. Overall, a total of 147 alleles were detected using the 16 microsatellite loci. The average number of alleles per locus was 7.52 (0.49), 7.35 (0.54), 6.3 (0.44), and 6 (0.38) for the Arab-Barb, Barb, Arabian, and English Thoroughbred breeds, respectively. The observed heterozygosities ranged from 0.63 (0.03) in the English Thoroughbred to 0.72 in the Arab-Barb breeds, whereas the expected heterozygosities were between 0.68 (0.02) in the English Thoroughbred and 0.73 in the Barb breeds. All F_ST values calculated by pairwise breed combinations were significantly different from zero (p  <  0.05) and an important genetic differentiation among breeds was revealed. Genetic distances, the factorial correspondence, and principal coordinate analyses showed that the important amount of genetic variation was within population. These results may facilitate conservation programs for the studied breeds and enhance preserve their genetic diversity.