Abstract: Background: The introduction of novel CTCF binding sites in gene regulatory regions in the rodent lineage is partly the effect of transposable element expansion, particularly in the murine lineage. The exact mechanism and functional impact of evolutionarily novel CTCF binding sites are not yet fully understood. We investigated the impact of novel subspecies-specific CTCF binding sites in two Mus genus subspecies, Mus musculus domesticus and Mus musculus castaneus, that diverged 0.5 million years ago. Results: CTCF binding site evolution is influenced by the action of the B2-B4 family of transposable elements independently in both lineages, leading to the proliferation of novel CTCF binding sites. A subset of evolutionarily young sites may harbour transcriptional functionality as evidenced by the stability of their binding across multiple tissues in M. musculus domesticus (BL6), while overall the distance of subspecies-specific CTCF binding to the nearest transcription start sites and/or topologically associated domains (TADs) is largely similar to musculus-common CTCF sites. Remarkably, we discovered a recurrent regulatory architecture consisting of a CTCF binding site and an interferon gene that appears to have been tandemly duplicated to create a 15-gene cluster on chromosome 4, thus forming a novel BL6 specific immune locus in which CTCF may play a regulatory role. Conclusions: Our results demonstrate that thousands of CTCF binding sites show multiple functional signatures rapidly after incorporation into the genome.