Hexokinases (HXK, Ec18.104.22.168), a key rate-limiting enzyme of glycolytic pathway, catalyze hexoses phosphorylation. HXKs not only help hexoses metabolism, but also play an important role in sugar signaling and tolerance to abiotic stress in higher plants. In past years, a number of HXK gene families have been reported in many plant species. However, detailed information about the HXK genes in sorghum is still lacking. To investigate the roles of HXKs in Sorghum bicolor, 7 putative sorghum HXK genes (SbHXK1, SbHXK2, SbHXK3, SbHXK4, SbHXK5, SbHXK6, SbHXK7) were identified in sorghum genome. Gene sequence analysis, structural analysis, phylogenetic analysis, collinearity analysis, multi-sequence alignment and motif analysis of all SbHXKgenes showed evolutionarily conservation to those in other plant species. Expression profiling revealed the potential activation of SbHXKs in ripping organisms, stamen and root tissues. Cis-elements related to abiotic stress and sucrose metabolism were identified through promoter screening in all SbHXKs, and further qRT-PCR confirmed the potential role of SbHXK genes in response to abiotic stress and sugar metabolism in sorghum. Interestingly, SbHXKs are mostly localized in cytoplasm, while some rice and ArabidopsisHXKs are localized in chloroplast or mitochondrion, which indicated the different role of SbHXKs comparing with other plant species. Altogether, our study shows a theoretical reference of HXK genes in Sorghum, which provides basic information that will assist in elucidating the functions of HXK gene family in Sorghum.