The conservation of genetic diversity in historically structured horse breeds requires fi-ne-scale population genetic evaluation beyond conventional pedigree-based approaches. The present study assessed genetic diversity, population structure, and mare family differentiation in a Lipizzan horse population using 16 ISAG/FAO-recommended microsatellite markers. A total of 172 mares representing 29 mare families were genotyped. All loci were polymorphic, with a mean number of alleles per locus of 6.69 and a mean effective number of alleles of 3.56. The average polymorphism information content (PIC = 0.66) indicated high marker informativeness. Mean observed heterozygosity was slightly lower than expected heterozygosity, although the overall negative inbreeding coefficient suggested general heterozygote excess and low population-level inbreeding, whilst the genetic differentiation among mare families was moderate. The principal components and the STRUCTURE analysis indicated admixture among mare families. Seven private alleles were detected across six loci, highlighting the presence of rare lineage-specific variants. These findings emphasize the importance of mare family-based analyses for identifying hidden genetic structures that may not be captured by population-level averages. The integration of microsatellite-based diversity evaluation provides a robust framework for de-signing sustainable breeding and conservation strategies aimed at preserving both overall variability and rare lineage-specific genetic components.

Lipizzan horses, bred for over four centuries, represent a unique genetic, cultural and historic resource. This study assessed genetic diversity and population structure across seven European Lipizzan populations, with emphasis on the Bosnia and Herzegovina population. A total of 547 Lipizzan horses were genotyped using 12 microsatellite markers. The parameters of genetic variability, admixture level and migration rate were calculated. The mean number of alleles, effective number of alleles and Shannon’s information index were 5.78, 3.24 and 1.31, respectively. The average fixation indices were estimated to be F_IT = 0.05 (SE = 0.02), F_IS = −0.02 (SE = 0.01), and F_ST = 0.07 (SE = 0.01). The level of genetic diversity observed within each Lipizzan horse subpopulation shows similar fluctuations, with the lowest values observed in Bosnia and Herzegovina and the highest in the Slovak Lipizzan population. The observed heterozygosity was lower than the expected heterozygosity. The Hardy–Weinberg equilibrium was maintained in Serbia, while minor deviations occurred in other populations, most notably in Slovakia. Nei’s genetic distances were highest between Slovakia and other populations, particularly Bosnia and Herzegovina. The population structure analysis confirmed admixture involving Bosnia and Herzegovina, and the migration pattern corresponded to geographic proximity and breeding practices. These findings provide a valuable basis for developing breeding strategies to reduce inbreeding and preserve genetic diversity.