Abstract: Two strains of Flammulina filiformis were used to create protoplast monokaryons which mating types were determined by mating test.The full compatiable di-mon mating was carried out, and the parents, hybrids and their protoplast monokaryons were used to ISSR-PCR spectrum analysis in order to provide reference for the identification of di-mon hybrid of Flammulina filiformis.The results showed that two types of protoplast monokaryons were obtained for both strains of FC6 and F93 while the ratio of two mating types for monokaryons were different.The hybrid strain of FC693 was obtained by the full compatiable di-mon mating of parent strains of FC6 P23 and F93.ISSR spectrum analysis showed that the specific fragment named T3 was detected in both FC693 and FC6 P23 while the specific fragment named T9 was detected in both FC693 and F93.These specific fragments were designed by SCAR primers and used to multiplex PCR for hybrid and parent strains.These ISSR-SCAR markers could distinguish the di-mon hybrid accurately and rapidly.