A total of 57 498 non-redundant ESTs were identified from 210 878 ESTs of Sorghum in NCBI by sequence analysis. In all, 3 338 SSRs were distributed in 3 116 ESTs with an average frequency of one SSR per 11.28 kb, which included 215 SSR motifs. Analysis of SSR motifs revealed that the trinucleotides were major motifs, accounting for 68.33%.The dinucleotides motifs accounted for 17.97%. There were 1 694 sequences from 3 338 EST-SSR sequences could be designed into primers and the proportion was 50.75%. Fourteen primers were selected to amplify EST-SSR loci with 50 collections of Sorghum bicolor × S. sudanenes, 7 collections of S. bicolor and 3 collections of S. sudanenes. Seventy-two allele variations were detected and the frequency was 5.14 gene loci per primer. The polymorphism index of each primer was in the range of 0.54-0.93. The genetic distance ranged from 0.1646 to 0.6398. This showed abundant genetic diversity in the materials. The materials were divided into 5 groups with clustering analysis of EST-SSR data. Each group included the varieties with similar parents or similar regional distribution. Meanwhile, 4 specific molecular markers were found. Primer D1763 was specific in the registered variety GB-4-2 which was the progeny of S. bicolor 314A × S. sudanenes White Skull. The marker was specific in justification of the germ difference. These results showed that the EST-SSR was an effective marker for genetic diversity analysis and specificity studies on S. bicolor × S. sudanenes.