Abstract: Using 3 different organs of root, stem and leaf of Sophora japonica cultivar 'Qingyun 1' as materials, the transcriptome was sequenced by high-throughput sequencing technology, and the differentially expressed genes in root, stem and leaf were screened for bioinformatics analysis and EST−SSR analysis. The results showed that there were 51937, 52711 and 7193 differentially expressed genes in root vs stem, root vs leaf and stem vs leaf, respectively. Further enrichment analysis of GO and KEGG showed that the differentially expressed genes were significantly enriched in Metabolic process, catalytic activity, glycolysis and glyconeogenesis. EST−SSR analysis showed that the length of SSR sequences in roots, stems and leaves of S. japonica ranged from 10 bp to 80 bp, mainly 10–22 bp and single nucleotide, 9 pairs of primers with high polymorphism were selected from 30 pairs of effective primers, and the fingerprints of 11 different clones of S. japonica were constructed.