In this review of methods for purity assessment of isolated chromosome X- and Y-bearing sperm, we compared the principles, operating procedures, as well as pros and cons for various methods. We conclude that nested PCR of single sperm will become a conventional and popular method with lower costs, and the method will play a very important role in optimizing the X, Y sorting method, if the sensitivity and accuracy of the method can be increased and the testing time decreased, and promote the new progress in other genetic testing techniques on single sperm.