Abstract: 5 pairs of bovine male-specific PCR primers were designed based on the sequences of bovine Y-specific repeat sequence, testis-specific protein gene on Y chromosome and sex determining region Y gene (SRY) , meanwhile, based on skeletal alpha action precursor gene and bovine 1.715 satellite DNA, 4 pairs of bovine-specific PCR primers were designed as internal control primers. After PCR amplification of bovine genomic DNA with different primers, 4 pairs of bovine male-specific primers and 1 pair of bovine-specific primers could work very well. Two primer combinations of bovine male-specific PCR primers and internal control primers, B34/A12 and B78/A12, were obtained for sexing bovine preimplantation embryo after multiplex PCR amplifications of bovine genomic DNA, fibroblasts, and clone embryos.