The creatine/phosphocreatine system is the principal energy buffer in mammals, but is scarcely documented in fish. We measured the gene expression of major enzymes of this system, glycine amidinotransferase (GATM), guanidinoacetate N-methyltransferase (GAMT) and muscle-type creatine kinase (CKM) in kidney, liver, and muscle tissues of fish and mammals. CKM was expressed strongly in the muscles of all examined species. In contrast, GATM and GAMT were strongly expressed in the muscle tissue of fish, but not of mammals. This indicates that creatine synthesis and usage are spatially separated in mammals, but not in fish, which is supported by RNA-Seq data of 25 species. Differences in amino acid metabolism along with methionine adenosyltransferase gene expression in muscle from fishes but not mammals further support a central metabolic role of muscle in fish, and hence different organization of the creatine/phosphocreatine biosynthesis system in higher and lower vertebrates.