Background and objectives: The DMRT3 gene, often referred to as the “gait keeper,” plays a key role in controlling alternative gaits in horses, such as t¨olt and pace. This study aimed to determine the frequency of known and to screen for potential novel polymorphisms within the second exon of the DMRT3 gene. Data collection: A total of 244 blood or hair samples were collected from representative individuals of the six horse breeds: gaited (Icelandic Horse, French Trotter), non-gaited (Arabian Horse, Malopolski Horse), and pony breeds (Welsh Pony, Shetland Pony). Research methods: The second exon of the DMRT3 gene analyzed using Sanger sequencing. Detected poly- morphisms were annotated and classified using Ensembl and NCBI databases. Allele frequencies and genotype distributions were statistically compared between breeds and breeding populations. Putative novel variants were further analysed using in silico approaches to predict their potential structural and functional consequences at the protein level. Results: Six polymorphisms were identified, including three novel variants. The known stop-gain variant c.902C>A (DMRT3_Ser301STOP) was confirmed in Icelandic Horses and French Trotters, with significant ge- notype differences between two French Trotter studs (p < 0.05). A novel 18-nucleotide in-frame duplication (c923_943dup; Ala297_Ala302dup) was found exclusively in Shetland Ponies. Additionally, a missense variant (c.967T>A; p.Tyr323Asn) and a synonymous change (c.855T>C) were detected in non-gaited breeds. In silico analyses suggested no major structural impact of the identified variants on the predicted DMRT3 protein. Contributions: This study confirms the distribution of the DMRT3_Ser301STOP allele in gaited breeds and iden- tifies novel polymorphisms in DMRT3. It expands current knowledge by identifying population- and breed- specific variants, suggesting a broader genetic influence on locomotor traits beyond gaited horses. The find- ings support the continued identification of DMRT3 as a molecular marker in equine locomotion research and selective breeding