Initiation and timely completion of DNA replication is an essential cellular function that must occur with high-fidelity, whist simultaneously navigating the structures imposed by the complex chromatin environment. Three-dimensional (3D) genome structure regulates a myriad of DNA functions, but despite major recent advances understanding how it impacts gene expression, it remains unclear how chromatin conformation participates in DNA replication initiation signalling and the subsequent dynamics of DNA polymerization. To address these unknowns, we intersected Hi-C (chromatin structure), Okazaki fragment sequencing (replication initiation and dynamics), and Repli-Seq (replication timing) in human primary and cancer cell lines. We found replication initiation is significantly enriched at TAD boundaries, specifically in early and mid S-phase. Strong replication fork directionality from TAD boundaries could occur in a bi- or uni-directional manner, which highly correlated with replication timing. Further, we observed evidence of strong uni-directional initiation zones arrayed in such a manner to promote overall uni-directional replication in mid and late S-phase. While TAD boundaries were largely invariant across cell lines, the distinct cell types shared a minority of initiation zones, indicative of cell type specific regulation. Despite these differences in replication initiation zones, live cell imaging demonstrated the spatiotemporal pattern of replication remained the same. Our findings provide new understanding of how 3D chromatin shapes the dynamics of DNA replication initiation and directionality. This is important because challenges to DNA replication (i.e. ‘replication stress’) is the primary driver of oncogenic genome instability. Hence targeting 3D chromatin structure to sensitize tumours to endogenous or therapeutically induced replication stress is a promising strategy for cancer therapy.