Hexokinase (HXK) catalyses hexose phosphorylation and senses glucose signalling to control carbon partition in plants. In sugarcane, HXK is involved in regulating source/sink carbon balance. However, the characteristics of sugarcane HXK genes remain unclear. This study revealed that the full-length cDNA sequence of the HXK gene ShHXK8 cloned from sugarcane mature leaves contains a 1560 bp open reading frame and encodes a putative protein of 519 amino acids. Amongst HXKs from monocot and dicot subjected to phylogenetic analysis, ShHXK8 was clustered into a monocot-specific group with OsHXK7, SbHXK8 and ZmHXK1. Gene expression analysis in Saccharum spp. hybrid ROC22 plants showed that ShHXK8 was expressed in leaves and stalks and expressed highly in mature leaves and young internodes. The difference in the expression of ShHXK8 was compared in high-sugar-content (HSC) and low-sugar-content (LSC) Saccharum officinarum plants. The expression of ShHXK8 in the leaves and internodes 3–24 of HSC plants was lower than that of LSC plants. The expression of ShHXK8 in the internodes 1–2 of HSC plants was higher than that of LSC plants. ShHXK8–GFP fusion was located in the cytosol by transiently expressing the corresponding vectors in rice mesophyll protoplasts. ShHXK8 might function as a cytoplasmic hexokinase involved in hexose metabolism in sugarcane mature leaves and young internodes.