Accurate replication and segregation of chromosomal DNA is essential for high-fidelity transmission of genetic information from generation to generation. Eukaryotic cells typically replicate by first duplicating their chromosomes during the S phase followed by their segregation between two daughter cells during the M phase. Over recent years, advances in our understanding of this process at the molecular level have been incredibly rapid. The present review will focus on molecular control of DNA replication and the mechanisms which operate to ensure that once replicated, chromosomes are not rereplicated in the same cell cycle.