Abstract: Nowadays, limited molecular markers were developed in Saccharum spontaneum. To increase the number of molecular markers of Saccharum spontaneum, in this study, the transferability of molecular markers from sorghum to Saccharum spontaneum was analyzed. 29 pairs of primer of genomic- SSR and 20 pairs of primer of EST-SSR from sorghum were screened in Saccharum spontaneum germplasm GSM39, to evaluate the transferability. Polymorphism and genetic diversity of the markers were further evaluated by using 14 materials of Saccharum spontaneum. The results showed that 70% EST- SSR primers were successfully amplified in Saccharum spontaneum, the percentage of polymorphism of available EST- SSR primers was 50% . The transferability rate of SSR primers in Saccharum spontaneum was only 34.5% , but the percentage of polymorphism was 70% . 14 polymorphism primers detected 33 alleles in 14 Saccharum spontaneum. The average observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He) was 2.4286, 1.7, 0.544 and 0.3858. Average value of Nei's genetic diversity index (H) was 0.3716. These results indicated that EST- SSR had higher transferability than that of SSR, but the percentage of polymorphism of SSR primers was higher. The research was significant for the study of genetic diversity and comparative genomics research in Saccharum spontaneum.