In this experiment,a L₁₆（4⁵） orthogonal design was used for the optimal SCoT-PCR reaction system with 24 Niuxinshi and 22 Wild persimmon accessions from National Field Genebank for Persimmon.The optimal PCR reaction mixture of SCoT-PCR system was 2.0 mmol/L Mg²⁺,0.2 mmol/L dNTPs,1.0 U Taq polymerase,0.5 μmol/L primer and 80 ng template DNA in 20 μL mixtures.Furthermore,the annealing temperature of each primer was optimized,to explore relative identification and variety classification of persimmon with SCoT markers.The results showed that:(1)A total of 176 loci were detected with 10 SCoT primers in 46 accessions,170 of which were polymorphic with a polymorphism of 96.6%.The genetic similarity ranged from 0.619 to 0.852.(2)The cluster analysis results showed that the 46 materials could be completely separated by using SCoT markers,Yemaoshi with staminate red flower and pistillate red flower were gathered for a group,they have far relationships with Niuxinshi group and Wild persimmon group,which is consistent with the morphologic observation.Bishan Niushishi was clustered on Wild persimmon group,which was based on its morphological characteristics showed the same as wild types.Bishan Niushishi is tetraploid,different from others which are hexaploid.Bishan Niuxinshi should belong to the Wild persimmon.The research indicated that SCoT markers can be effectively used to evaluate the genetic diversity of persimmon germplasm.46 persimmon accessions have high genetic diversity,reflecting the geographical distribution of species to a certain extent,and also show geographical factors have a significant impact on the genetic relationship for persimmon.