The Kustanay horse is a riding breed developed in Kazakhstan whose genetic diversity and differentiation remain insufficiently studied. We assessed genetic diversity and population structure using 16 autosomal STR loci from a 17-locus panel comprising 12 ISAG core markers. Of 765 genotyped horses, 703 remained after excluding duplicate records and close relatives; the X-linked LEX3 locus was excluded from population analyses. Five groups were studied: registered Kustanay (KST, n = 32), Kustanay from Qazaq Tulpary (KT, n = 36), Thoroughbred (TB, n = 31), Kustanay crossbred (CROSS, n = 9), and undocumented origin (UB, n = 595). Expected heterozygosity was high across groups (H_E = 0.661–0.725). Overall F_ST was 0.0088, and AMOVA attributed 99.13% of molecular variance to variation within individuals. The Puechmaille estimators favored K = 2, whereas Evanno ΔK peaked at K = 4; neither solution revealed clear separation among groups. Nei’s genetic distances and PCoA were consistent with weak differentiation and similarity between registered Kustanay and undocumented horses. Unequal group sizes limit interpretation, and these findings do not establish breed identity for individual undocumented horses. The results inform pedigree recording and conservation planning.