[Objective] This study discussed the SNPs of SLC27A1 gene and its relationship with milk production traits in Chinese Holstein Cattle in order to find the SNP site which had significant effect on milk production traits in Chinese Holstein Cattle.[Method] DNA was extracted from the bleed of 48 Chinese Holstein Cattle selected according to phenotypic character and mixed into DNA pool for SNPs detection by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP)and cloning sequencing.Then different genotypes were detected in other 231 Chinese Holstein Cattle by PCR-RFLP.The association between genotype and production traits was assessed by GLM procedure,SAS version 8.02.[Result] There were T112C in exon3 and G64A loci in 3'UTR,among them the T112C in exon3 was synonymous mutation.There were 3 genotypes TT,TC and CC in T112C locus and 3 genotypes GG,GA and AA in G64A locus.The population was at Hardy-Weinberg equilibration.Cows with genotype CC had significantly highest milk yield than those with genotype TC(P0.01),and there were no significant differences among the 3 genotypes on milk protein percent and milk fat percent(P0.05),but the tendency of CC TC TT on milk protein percent and the tendency of TT TC CC on milk fat percent were showed.There was no significant difference among the 3 genotypes of G64A loci on milk yield,milk protein percent and milk fat percent(P0.05),but the tendency of GA GG AA on milk yield and the tendency of AA GG GA both on milk protein percent and milk fat percent were showed.[Conclusion] There was certain relation between the T112C locus and milk yield traits;It may improve milk yield to raise the frequency of genotype CC;SLC27A1 gene could be a useful candidate gene in selection program on milk yield traits in Chinese Holstein Cattle,which provided a theoretical basis for the marker-assisted breeding and further study of SLC27A1 gene.