Abstract: Current invention is about the sex-specific separation of sperm in high purity. For more details, this invention is about the methods for improving the separation efficiency of sperm by removing factors deteriorating separation efficiency by utilizing several parameters and adjusting few conditions for the purpose of separating sperm into X-chromosome bearing and Y-chromosome bearing groups in high purity. Current invention, for the purpose of separating X-chromosome bearing and Y-chromosome bearing sperm based on physiological characteristics, utilize the difference of sperm's nucleus width as a parameter to recognize the difference in DNA content between X and Y chromosome. Current invention is about the sex-specific separation of sperm in high purity.