The enzyme glucokinase (GK) (EC 2.7.1.1) plays an important role in the control of glucose homeostasis. Qualitative and/or quantitative variations in GK enzyme have been postulated by previous studies to explain why dietary carbohydrate utilisation is lower in gilthead seabream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss) than in common carp (Cyprinus carpio). In this study, we report the isolation and characterisation of a full-length cDNA coding for GK in these teleosts. Amino acid sequences derived from these cDNA clones are highly similar to other vertebrate GKs. These findings, including a detailed phylogenetic analysis, reveal that GK gene highly homologous to mammalian GK exists in these fish species with similar tissue specific expression (mainly liver).