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 de-creased, and promote the new progress in other genetic testing techniques on single sperm.