Abstract: Soybean is an important economic crop in the world. However, Soybean is often subjected to various stresses during its growth and development, which seriously affects its yield and quality. As a member of the heat shock protein family, small heat shock protein（Hsp20） plays an important role in protecting plants from abiotic stress. In order to explore the characteristics of soybean Hsp20 family genes and their responses to high temperature and humidity stress, the whole genome analysis was carried out, and the evolutionary relationship, conservative domain, chromosome position, promoter cis regulatory elements and tissue specific expression were predicted and analyzed using bioinformatics methods. The protein expression of Xiangdou 3 and Ningzhen 1 soybean varieties under high temperature and high humidity stress at different times was analyzed by using isotope labeled relative and absolute quantification（iTRAQ） techniques. The results showed that 33 GmHsp20 genes were identified in soybean genome, distributed unevenly on 16 chromosomes and divided into 11 subgroups according to phylogenetic analysis and subcellular localization results. Sequence analysis showed that the GmHsp20 protein was highly conserved in structure and contained a typical ACD domain. Most of the GmHsp20 genes（96.96%） had no introns or only one intron. Promoter analysis showed that the GmHsp20 gene contained a large number of cis acting elements related to stress, with the largest number of MYBs（110）. The prediction of tissue specific expression of genes showed that all genes except GmHSP22.6 were expressed in various tissues and organs. Among them, GmHSP18.5, GmHSP23.7 and GmHSP15.2 were highly expressed in all tissues. Quantitative proteomics results showed that GmHSP18.5, GmHSP23.7, GmHSP26.0B, GmHSP17.8 and GmHSP25.9 were up-regulated and expressed in both Ningzhen 1 and Xiangdou 3 varieties after high temperature and high humidity stress treatment compared to the control, and the expression trends were essentially the same between the two varieties. This suggests that these members of the small heat shock protein family might participate in the response process of soybean to heat and humidity stress. The results of this study provide valuable information for further investigation of GmHsp20 function.