【Objective】Based on the data of transcriptome sequencing, 200 pairs of SSR primers were detected using six proso millet accessions with different origin. The aim of this study is to construct a set of molecular markers for assessing the genetic diversity of proso millet germplasms.【Method】Primer Premier 5.0 software was used to design primers, DNA was extracted by modified CTAB method and amplified by PCR. Polyacrylamide gel electrophoresis was used to screen the polymorphism of primers. Genetic diversity parameters were calculated with PowerMarker 3.25 and PopGen 1.32.【Result】Ninety seven and eighty out of 200 pairs of primers were shown monomorphic and polymorphic, respectively. A total of 20 were mono-nucleotide SSRs, among them 10 were polymorphic with repeat motif A (50%) and T (50%). A total of 36 were di-nucleotide SSRs, among them 15 were polymorphic with seven types of repeat motif, AG class was the most frequent, followed by the TC, GC and GA, CA, TA and AC were the least. A total of 144 were tri-nucleotide SSRs, among them 55 were polymorphic with 24 types of repeat motif, GGC, GCG and GCC class were the most frequent, followed by the GAA, GCT, CGC, etc, ACC, AGG, CAG, CGT, AAG, AAC, TCG, CGA, ATT, CAA and CCA were the least. In terms of the mono-, di-, tri-nucleotide SSR, their Rp value ranged from 0.67 to 4.67 (mean 2.07), 1.33 to 4.33 (mean 2.73) and 0.67 to 4.00 (mean 1.83), respectively. Evaluating the distribution frequency based on Rp, 80 SSRs were divided into five intervals viz 0-1, 1-2, 2-3, 3-4 and 4-5, with 17 (21.25%), 36 (45.00%), 11 (13.75%), 14 (17.50%) and 2 (2.50%) markers, respectively. For the mean of the Rp value, the mono-, di-, tri-nucleotide SSR were 0.33 to 0.67 (average 0.51), 0.40 to 0.78 (average 0.59) and 0.33 to 0.83 (average 0.59), respectively. As for mono-nucleotide SSRs, a total of 22 allelic variations were detected with 2-3 alleles (average 2.2000) for each locus; 2 and 3 alleles were identified for 8 and 2 loci, respectively. In terms of di-nucleotide SSRs, a total of 38 allelic variations were generated with 2-3 alleles (average 2.5333) for each locus; 7 and 8 loci produced 2 and 3 alleles, respectively. As for tri-nucleotide SSRs, a total of 136 allelic variations were generated with 2-3 alleles (average 2.4727) for each locus; 29 and 26 loci produced 2 and 3 alleles, respectively. In terms of the polymorphism information content, the mono-, di-, tri-nucleotide SSR were 0.3750-0.5355, 0.2392-0.7438 and 0.2392-0.7438, respectively. In terms of the diversity index, the mono-, di-, tri-nucleotide SSR were 0.6365-1.0776 (average 0.7497), 0.5623-1.0986 (average 0.8339) and 0.5623-1.0889 (average 0.8312), respectively. 【Conclusion】Two hundreds pairs of SSR primers obtained from transcriptome sequencing were used to detect genetic diversity of 6 proso millet accessions. It was shown that 177 (88.5%) out of the 200 pairs primers amplified intact bands, and 80 SSRs appeared polymorphic with the polymorphic rate 40%.