Abstract: In order to identify the key genes responsive to drought stress and to elucidate the underlying mechanism in drought tolerance of soybean seedlings, Zhechun 3 and its mutant Gm-lpa-ZC-2（ZC-2） were test in this research. The phenotype, relevant physiological and biochemical indexes were observed and determined after stress treatment under 20% PEG6000, and RNA-seq technology used for transcriptomic analysis. The results showed that the relative water content, the maximum quantum yield of PSII（Fv/Fm）, soluble sugar content and soluble protein content of the mutant ZC-2 were significantly higher than those of Zhechun 3, and relative electrolyte leakage rate（REC） and malondialdehyde（MDA） content were lower than those of Zhechun 3, which could indicate that the mutant ZC-2 was more drought tolerant than Zhechun 3. The transcriptomic data indicated that there were totally 7 879 differentially expressed genes（DEGs） between Zhechun 3 and ZC-2, including 4 208 up-regulated genes and 3 671 down-regulated genes. There were 571 differential genes expressed on both day 2 and day 4 under treatment. In addition, the 571 DEGs were involved in 84 metabolic pathways. KEGG enrichment analysis indicated that DEGs were mainly enriched in pathways such as photosynthesis-antenna proteins, galactose metabolism, protein processing in endoplasmic reticulum, and flavonoid biosynthesis. Moreover, genes related to photosynthesis and sugar metabolism pathway as well as gene associated with drought stress, including TIFY10A, ZAT10, RD22, PM19L and XTH6, were detected. This study provided a foundation for mining candidate genes related to drought tolerance in soybean seedlings and studying the mechanism of soybean drought tolerance.