Exposure to a temperature increase may disrupt smoltification and delay or stop the downstream migration of smolts. Thermal regimes are often different between a river and its tributaries, but the effects of a relative temperature shift are not well described. We used expression of smoltification genes coupled with gill Na+/K(+)ATPase activity (NKA) and plasma cortisol and growth hormone (GH) levels to investigate the impact of a 5 degrees C difference between tributary and river on salmon juveniles. Responses to a temperature challenge were examined at four time points during the smoltification period, with juveniles reared under three regimes including control, early and late temperature increase. The temperature shifts reduced gill NKA, plasma GH and cortisol levels which indicate hypo-osmoregulation impairment and may reduce the survival of smolts. Out of the 22 genes examined, the expression of six genes was influenced by the temperature treatments, while changes in further eleven genes were influenced by the date of sampling. Genes usually known to be upregulated during smoltification were downregulated after the temperature increase, notably nkaa1b, nkcc1a and igf1r. Upregulation of some genes involved in the hormonal regulation and acid-base equilibrium in early June may indicate a switch towards desmoltification. This study gives further insights about the impact of temperature increase on the molecular processes underlying smoltification and possible responses to human-related water temperature increase. The data also suggest dual roles in the smoltification and desmoltification for GH and IGF1 and points to the implication of genes in the smoltification process, that have previously been unstudied (nbc) or with little data available (igf2).