Laboratory experiments were undertaken to ascertain whether or not individuals of two allopatric Otomys irroratus populations (Kamberg and Karkloof) distinguish between mates from the same (homotype) and the other (heterotype) population. The study aimed to establish the existence of pre-mating reproductive isolation mechanisms between the two populations. In both ‘whole-animal choice’ trials and olfactory discrimination tests, all females significantly preferred homotype stimulus males or odour. Although males of both populations preferred homotype females, males were less discriminating at the start of experiments, possibly because of increased exploratory drive at this time. Furthermore, Kamberg males demonstrated equal preference for both stimulus females during the dark phase of the light cycle; no explanation is given for this phenomenon. Overall, the results indicate the existence of population-specific courtship behaviour, with olfactory cues apparently playing an important role in mate recognition. It is therefore possible that, should representatives of the two populations meet in nature, they may be reproductively isolated through behavioural means.