Abstract The 3D genome is essential to numerous key processes such as the regulation of gene expression and the replication-timing program. In vertebrates, chromatin looping is often mediated by CTCF, and marked by CTCF motif pairs in convergent orientation. Comparative Hi-C recently revealed that chromatin looping evolves across species. However, Hi-C experiments are complex and costly, which currently limits their use for evolutionary studies over a large number of species. Here, we propose a novel approach to study the 3D genome evolution in vertebrates using the genomic sequence only, e.g . without the need for Hi-C data. The approach is simple and relies on comparing the distances between convergent and divergent CTCF motifs (ratio R ). We show that R is a powerful statistic to detect CTCF looping encoded in the human genome sequence, thus reflecting strong evolutionary constraints encoded in DNA and associated with the 3D genome. When comparing vertebrate genomes, our results reveal that R which underlies CTCF looping and TAD organization evolves over time and suggest that ancestral character reconstruction can be used to infer R in ancestral genomes.