Abnormal DNA replication is the primary way that cancer develops in mammals; therefore, a deep understanding of the way replication works for healthy cells will enhance our ability to eradicate problematic replication pathways. The same rapid advances in technology within the last ten to twenty years that have allowed us to understand DNA replication better have also led and will lead to new cancer therapies. In recent years, our understanding of the complexity of DNA replication has advanced tremendously. This e-book distills the bulk of the published studies in DNA replication with an intentional focus on eukaryotes, specifically, budding yeast and mammals. An important feature of this e-book is the incorporated images and figures. Being able to clearly visualize protein and enzymatic processes is central to understanding them. Therefore, we have incorporated images of the three-dimensional structures of the proteins that mediate DNA replication, stepwise guides to simplify the complex nature of the replication process, and cryo-EM images for different proteins and protein–DNA complexes to reveal their structural components. We hope to have provided readers with both fundamentals and cutting-edge information so that they may think about the biology of DNA replication and contribute to the body of knowledge in the field.