The J-protein family comprises molecular chaperones involved in plant growth, development, and stress responses. Little is known about this gene family in soybean. Hence, we characterized J-protein genes in soybean, with the most highly expressed and responsive during flower and seed development. We also revealed their phylogeny, structure, motif analysis, chromosome location, and expression. Based on their evolutionary links, we divided the 111 potential soybean J-proteins into 12 main clades (I–XII). Gene-structure estimation revealed that each clade had an exon-intron structure resembling or comparable to others. Most soybean J-protein genes lacked introns in Clades I, III, and XII. Moreover, transcriptome data obtained from a publicly accessible soybean database and RT-qPCR were used to examine the differential expression of DnaJ genes in various soybean tissues and organs. The expression level of DnaJ genes indicated that, among 14 tissues, at least one tissue expressed the 91 soybean genes. The findings suggest that J-protein genes could be involved in the soybean growth period and offer a baseline for further functional research into J-proteins' role in soybean. One important application is the identification of J-proteins that are highly expressed and responsive during flower and seed development in soybean. These genes likely play crucial roles in these processes, and their identification can contribute to breeding programs to improve soybean yield and quality.