Normal anxiety is an adaptive emotional response. However, when anxiety appears to lack adaptive value, it might be defined as pathological. Adaptation in animals can be assessed for example by changes in behavioural responses over time, i.e. habituation. We hypothesize that non-adaptive anxiety might be reflected by impaired habituation. To test our hypothesis, we repeatedly exposed male mice from two inbred strains to a novel environment, the modified hole board. BALB/cJ mice were found to be initially highly anxious, but subsequently habituated to the test environment. In contrast, 129P3/J mice initially showed less anxiety-related behaviour compared with the BALB/cJ mice but no habituation in anxiety-related behaviour was observed. Notably, anxiety-related behaviour even increased during the experimental period. Complementary, 129P3/J mice did not show habituation in other parameters such as locomotor and exploratory activity, whereas significant changes appeared in these behaviours in BALB/c mice. Finally, the expression of the immediate early gene c-fos differed between the two strains in distinct brain areas, known to regulate the integration of emotional and cognitive processes. These results suggest that 129P3/J mice might be a promising (neuro)-behavioural animal model for non-adaptive, i.e. pathological anxiety.