The activities of certain genes as a consequence of exposure to stressors related to typical handling procedures in aquaculture have not been studied in sufficient detail in fish. A stress trial with koi carp was, therefore, conducted, aiming at identifying relevant gene expression patterns in different brain regions during stress responses. The following stressors were selected: tank manipulation, distress caused by exposure to air and eustress due to feed rewards. Responses to these stressors were evaluated 10, 30 and 60 min after their application. The exact determination of gene expression profiles in the carp brain required the comparison of several suitable reference genes, which is also highly recommended for other studies focusing on the fish brain. Moreover, and as expected, the mRNA expression of a number of early immediate genes indicated activity in different brain regions as a response to changes in rearing and experimental conditions. In addition, the mRNA expression of metabolic genes was investigated, since increased brain activity may also increase the metabolic demands of certain brain areas. Furthermore, genes related to the stress axis were included in the study. The mRNA expression patterns of genes belonging to the stress axis revealed that negative stress caused by exposure to air had broad‐ranging effects on the gene regulation patterns in the fish brain, even if the fish were only treated for 1 min. This parallels the effects that have been observed on blood cortisol and glucose. In contrast, a limited number of genes allows discrimination of eustress and distress, which indicates that further research is needed in the future. Finally, the use of different control groups is highly recommended for fish experiments to correct for typical experimental procedures such as lid openings or social isolation.