Early life is an important period for the development of the nervous system and for the programming of behavioural phenotypes in adulthood. In our study, two types of early-life stress were used: prolonged separation of pups from their mothers (for 3 h/day, maternal separation (MS)) and brief separation (for 15 min/day, handling (HD)). We analysed the effects of early-life stress on behaviour and the expression of HPA-associated genes in the hypothalamus, hippocampus, and frontal cortex of male mice. Adult mice in the MS group demonstrated reduced locomotor activity and deficiencies in spatial long-term memory, while the HD showed no significant changes. Additionally, early-life MS resulted in reduced hippocampal Crhr1 mRNA, increased MR/GR mRNA in the hippocampus and hypothalamus. Both groups, HD and MS, showed increased Avp mRNA in the hypothalamus. Thus, prolonged maternal separation but not brief leads to adverse behavioural changes and influences the expression of HPA-associated genes in a brain region-specific manner.