This experiment aimed to investigate the polymorphism of the Acot2 gene in the grassland red cattle, providing reference data for the auxiliary selection of genetic markers in the breeding of grassland red cattle. In this experiment, 127 grassland red bulls were randomly selected as the research object, extracted blood DNA to amplify all exons of Acot2 gene, obtained the amplified fragment sequence by Sanger sequencing, obtained polymorphic sites through sequence alignment, and analyzed the polymorphism of Acot2 gene in grassland red cattle. The results showed that there was a mutation at the chr10:84971902 bp of Acot2 gene in grassland red cattle, the codon changed from ACG to GCG, at the mutation site, A was the dominant allele, AA was the dominant genotype, and the polymorphism information content showed that the mutation site of Acot2 gene belonged to low polymorphic locus. Missense mutations caused by base mutations lead to the conversion of threonine encoded by the original sequence to alanine,which may lead to structural changes of proteins.