The Doublesex and Mab-3 Related Transcription Factor 3 (DMRT3) gene encodes an important transcription factor in the setting of spinal cord circuits controlling movement in vertebrates. The SNP g.22999655C>A of the DMRT3 gene was significantly associated with performance of 4-beat gaits in horses, such as the running walk and the amble American Saddlebreds ridden and shown under saddle seat in both 3-gaited (walk, trot, and canter) or 5-gaited classes (walk, trot, slow gate, rack, and canter). We investigated whether SNP g.22999655C>A of DMRT3 was more prevalent among 5-gaited horses than among the random population of Saddlebred horses. The genotyping of the SNP of the DMRT3 gene was performed by PCR-RFLP using the restriction enzyme DdeI. The frequency of the A allele among 37 randomly selected Saddlebreds was 0.28. This is in agreement with results from a previous study in which the prevalence of the A allele in American Saddlebred horses was 27.5. Only 3 of the horses in our study had been shown as 5-gaited horses and all 3 had the genotype A/C. Although consistent with the hypothesis that the A allele is more prevalent among for 5-gaited horses, the numbers are too small for any conclusion. Additional Saddlebred horses, and especially 5-gaited horses, will be tested and results reported allowing determination of statistical significance.