Significant morphological and physiological variations between root vole Microtus oeconomus (Pallas, 1776) populations have been found. Behavioural differences between two geographically separated populations were found in this study. Ultrasonic vocalization and behavioural interaction (non-aggressive and aggressive approaches) were monitored during 10-min encounters. Ultrasounds were monitored during encounters involving all possible combinations of males and females of two populations, called here southern (Prague, Czech Republic) and northern (Białowieża, Poland). The northern male-male combination did not produce ultrasounds. In both populations, males exhibited more aggressive behaviour than females did. Since M. oeconomus females are highly territorial and the home ranges of males overlap, the agonistic behaviour of males may enable them to control a larger area with greater numbers of receptive females. We also found significant differences in behavioural activity between the two investigated populations: southern M. oeconomus showed higher activity than northern root voles did. Separate populations which differ morphologically and behaviourally have much to tell us about speciation processes. Behavioural divergence can lead to reproductive isolation and thus create separate gene pools.