DNA replication occurs in a defined spatiotemporal order, which is mediated by the firing of origins across the genome at different times during S phase. Replication timing is associated with gene regulation and influences mutation rates across the genome, but the evolutionary forces shaping replication timing are largely unknown. In this dissertation, I profiled DNA replication timing from humans, chimpanzees, and rhesus macaques to study the causes and consequences of replication timing evolution. I found that hundreds of regions vary in replication timing within and between the genomes of humans and chimpanzees, and over a hundred regions were classified as changes that occurred specifically in the human lineage. Importantly, human-chimpanzee variants highly overlap regions of within species variation, which points to the presence of ongoing evolution. Replication timing variation was also found to be correlated with regulatory evolution (e.g. gene expression and chromatin structure) and had elevated levels of sequence divergence. Finally, linking genetic variation to replication timing variation within humans and chimpanzees facilitated the identification of sequence determinants of replication timing evolution. Overall, DNA replication timing shows ongoing evolution in the human lineage, at least in part driven by sequence alterations, and with important implications for regulatory and sequence evolution.