Heat shock transcription factors (HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. In this study, a genome-wide analysis was carried out to identify all HSFs soybean genes. Twenty six nonredundant HSF genes (GmHsf) were identified in the latest soybean genome sequence. Chromosomal location, protein domain and motif organization of GmHsfs were analyzed in soybean genome. The phylogenetic relationships, gene duplications and expression profiles of GmHsf genes were also presented in this study. According to their structural features, the predicted members were divided into the previously defined classes A–C, as described in Arabidopsis. Using RT-PCR, the expression patterns of 26 GmHsf genes were investigated under heat stress. The data revealed that these genes presented different expression levels in response to heat stress conditions. Real-time (q)RT-PCR was performed to investigate transcript levels of five GmHsfs in response to multiple abiotic stresses. Differential expression of five GmHsfs implies their role during abiotic stresses. Subcellular localization using GFP-fusion protein demonstrated that GmHsf12 and GmHsf34 were restricted to the nucleus and GmHsf28 was localized in the nucleus and cytoplasm in plant. The results provide a fundamental clue for understanding of the complexity of the soybean HSF gene family and cloning specific function genes in further studies and applications.