Abstract: Based on the multiple base deletions on the fifth intron of the sheep AMELX/Y（sAMELX/Y）gene，a pair of specific primers was designed to amplify the different sequences of AMEL gene on X and Y chromosome of sheep blood, fibroblasts and embryos by two-temperature -PCR in this test. The results showed that a 280 bp fragment from the female sheep DNA（XX）was amplified, and two fragments（280 bp and 235 bp respectively）were got from the male sheep DNA（XY）. The accuracy rate of sex identification of 50 samples（25 females and 25 males）was 100%.