Rainbow trout have, as salmonid fish species, undergone sequential genome duplication events in their evolutionary history. In addition to a teleost-specific whole genome duplication approximately 320-350 million years ago, rainbow trout and salmonids in general underwent an additional salmonid lineage-specific genome duplication event approximately 80 million years ago. Through the recent sequencing of salmonid genome sequences, including the rainbow trout, the identification and study of duplicated genes has become available. A particular focus of interest has been the evolution and regulation of rainbow trout gluconeogenic genes, as recent molecular and gene expression evidence points to a possible contribution of previously uncharacterized gluconeogenic gene paralogues to the rainbow trout long-studied glucose intolerant phenotype. Since the publication of the initial rainbow trout genome draft, resequencing and annotation have further improved genome coverage. Taking advantage of these recent improvements, we here identify a salmonid-specific genome duplication of ancestral mitochondrial phosphoenolpyruvate carboxykinase 2 isoenzyme, we termed pck2a and pck2b. Cytosolic phosphoenolpyruvate carboxykinase (Pck1) and, more recently mitochondrial Pck2, are considered to be the rate-limiting enzymes in de novo gluconeogenesis. Following in silico confirmation of salmonid pck2a and pck2b evolutionary history, we simultaneously profiled cytosolic pck1 and mitochondrial pck2a and pck2b expression in rainbow trout liver under several experimental conditions known to regulate hepatic gluconeogenesis. Cytosolic pck1 abundance was increased by nutritional (diets with a high protein to carbohydrate ratio compared to diets with a low carbohydrate to protein ratio) and glucoregulatory endocrine factors (glucagon and cortisol), revealing that the well-described transcriptional regulation of pck1 in mammals is present in rainbow trout. Conversely, and in contrast to mammals, we here describe endocrine regulation of pck2a (decrease in abundance in response to glucagon infusion), and nutritional, social-status-dependent and hypoxia-dependent regulation of pck2b. Specifically, pck2b transcript abundance increased in trout fed a diet with a low protein to carbohydrate ratio compared to a diet with a high protein to carbohydrate ratio, in dominant fish compared to subordinate fish as well as hypoxia. This specific and differential expression of rainbow trout pck2 ohnologues is indicative of functional diversification, and possible functional consequences are discussed in light of the recently highlighted gluconeogenic roles of mitochondrial pck2 in mammalian models.