Objective To knock out the target chromosome by CRISPR/Cas9 system to cut multiple sites of Y chromosome, and provide a new method for sex control of livestock and poultry. Method Based on CRISPR/Cas9 technology, we searched for multiple copies of repeat sequences on Y chromosome that can be specifically recognized by sgRNA, and verified their effectiveness on target by in vitro cleavage, quantitative analysis and karyotype identification. Result The designed sgRNA could cut the target fragment obviously in vitro, and the cutting efficiency was more than 50%. The results of quantitative analysis of genes further proved the effectiveness of gene cutting at the cell level, and the cutting effect of clustered repeats was significantly better than that of scattered repeats. Karyotype identification also confirmed the loss of pig Y chromosome at the cellular level. Conclusion The research results lay a foundation for the subsequent construction of chromosome knockout pigs and the realization of sex control in pigs.