CCCTC-binding factor (CTCF) is a conserved architectural protein that plays crucialroles in gene regulation and three-dimensional (3D) chromatin organization. To bet-ter understand mechanisms and evolution of vertebrate genome organization, weanalyzed genome occupancy of CTCF in zebrafish utilizing an endogenouslyepitope-tagged CTCF knock-in allele. Zebrafish CTCF shares similar facets with itsmammalian counterparts, including binding to enhancers, active promoters andrepeat elements, and bipartite sequence motifs of its binding sites. However, wefound thatin vivoCTCF binding is not enriched at boundaries of topologically asso-ciating domains (TADs) in developing zebrafish, whereas TAD demarcation by chro-matin marks did not differ from mammals. Our data suggest that general mecha-nisms underlying 3D chromatin organization, and in particular the involvement ofCTCF in this process, differ between distant vertebrate species.