Abstract: cDNA of α-actinin1 gene was cloned by RT-PCR based on the total RNA from bovine testicle. Furthermore, the sequence of cDNA was analysed using bioinformatics. A1-bd pair of primers and β-actin pair of primers were used to amplify α-actinin1 gene based on the total RNA from 12 bovine tissues, including liver, testicle, lung, rumen, uterus, small intestine, spleen, heart, fat, ovary, kidney and muscle. In addition, mutation was detected by DNA sequencing and PCR-RFLP in twinning Luxi cattle, Luxi cattle, Nili-Ravi bufflo, Murrah buffalo, Jinnan cattle, Holstein, Simmental and Nanyang cattle. The result indicated that the cDNA sequence was 2 911 bp, which contained an open reading frame of 2 679 bases (from 53 to 2 731 bp) which encoded an 892 amino acid. The α-actinin 1 protein had 3 mainly structural domains including CH, SPEC and EFh. α-actinin1 gene was expressed in liver, testicle, lung, rumen, uterus, small intestine, spleen, heart, fat and kidney, there was no express in ovary and muscle. Mutation of A/G was detected in intron 13 of α-actinin1 gene by 1.5% gelose gel electrophoresis. The mutation caused Hae Ⅲ polymorphism in group, so there were AA and AG genotypes. The association analysis of this Hae Ⅲ polymorphism with reproduction traits showed that Hae Ⅲ PCR restriction fragment length polymorphism (RFLP) genotypes may be no association with the number of calving (P＞0.05). In conclusion, the cDNA of α-actinin1 gene was obtained. A mutation was detected in all populations besides Holstein. There was no association between the number of calving and genotypes.