The aim of this study was to establish whether two lines of rainbow trout divergent for their plasma cortisol response to a standardized stressor would show consistent differences in their behavioural response to a range of challenging situations. Our results show that the high- and low-responding (HR and LR) lines of rainbow trout did not differ in the aggression shown towards an intruder or in their response to the introduction of a novel object to their home environment. However, there was a difference in behaviour between the two selection lines when they were exposed to two unfamiliar environments. These results suggest that the behaviour of the HR and LR fish differs when they are challenged in unfamiliar environments, while their behaviour does not differ when they are challenged in their home environment. These observations are in agreement with studies on mammals that show that individuals with reactive coping styles perform similarly to proactive animals when they are challenged in a familiar environment, while they show different behaviour when they are challenged in unfamiliar environments. Thus, these results provide further evidence that the HR and LR selection lines of rainbow trout exemplify the two different coping styles described in mammals.