ABSTRACT Non-model rodents are widely used as subjects for both basic and applied biological research, but the genetic diversity of the study individuals is rarely quantified. University-housed colonies tend to be small and subject to founder effects and genetic drift and so may be highly inbred or show substantial genetic divergence from other colonies, even those derived from the same source. Disregard for the levels of genetic diversity in an animal colony may result in a failure to replicate results if a different colony is used to repeat an experiment, as different colonies may have fixed alternative variants. Here we use high throughput sequencing to demonstrate genetic divergence in three isolated colonies of Mongolian gerbil ( Meriones unguiculatus ) even though they were all established recently from the same source. We also show that genetic diversity in allegedly ‘outbred’ colonies of non-model rodents (gerbils, hamsters, house mice, and deer mice) varies considerably from nearly no segregating diversity, to very high levels of polymorphism. We conclude that genetic divergence in isolated colonies may play an important role in the ‘replication crisis’. In a more positive light, divergent rodent colonies represent an opportunity to leverage genetically distinct individuals in genetic crossing experiments. In sum, awareness of the genetic diversity of an animal colony is paramount as it allows researchers to properly replicate experiments and also to capitalize on other, genetically distinct individuals to explore the genetic basis of a trait.