Abstract: [Objective] In this study,the project aims to analyze the association of DMRT3 gene polymorphism with gait trait in trotting type of 103 individuals Yili horse.[Method] Genetic polymorphism of DMRT3 gene was detected by PCR-RFLP and DNA sequencing technology.[Result] The result showed that there was 1 mutation in DMRT3 gene and there were 3 genotypes:CC,CA and AA.The SNP g.22999655 C ＞ A was identified in DMRT3 gene by sequencing,and it caused the premature stop of the encoded acid.Statistical results indicated that the SNP was significantly associated with speed in Yili horse (P ＜ 0.05).[Conclusion] The mutation might be acted as an crucial DNA genetic marker for trotting performance traits selection in Yili horse.