The enzyme carnitine palmitoyltransferase (CPT) I is a major regulator of mitochondrial fatty acid oxidation in vertebrates. Numerous genome duplication events throughout evolution have given rise to three (in mammals) or multiple (in fish) genetically and functionally different isoforms of this enzyme. In particular, these isoforms represent a diversification of kinetic and regulatory properties stemming from mutations at the genomic and proteomic levels. Phylogenetic reconstructions reveal a comprehensive view of the CPT I family in vertebrates and genomic modifications leading to structural changes in proteins and functional differences between tissues and taxa. In a model fish species (rainbow trout), the presence of five CPT I isoforms suggests repeated duplication events in bony fishes and salmonids. Subsequently, an array of nucleotide and amino acid substitutions in the isoforms may contribute to a tissue-specific and a previously observed species-specific difference in the IC50 for malonyl-CoA. Moreover, all five isoforms are expressed in trout at the mRNA level in skeletal muscle, heart, liver, kidney, and intestine. In general, transcript levels of the β-isoforms were higher in muscle tissues, while levels of the α-isoforms were higher in other tissues. Rainbow trout also exhibit developmental plasticity in relative mRNA expression of CPT I isoforms from fry to juvenile to adult stage. Thus the evolution of CPT I has resulted in a very diverse family of isoforms. These differences represent a degree of specificity in the ability of species to regulate function at the protein and tissue levels, which, in turn, may allow for precise control of lipid oxidation in individual tissues during physiological perturbations.