The eukaryotic genome is replicated according to a tightly regulated temporal program that ensures each DNA segment is copied once per cell cycle. This program reflects the coordinated action of replication origin licensing, chromatin state, transcriptional activity, and nuclear organisation. While the core mechanisms of licensing and initiation are well characterised, the determinants of origin selection and firing time remain incompletely understood. In budding yeast, origins are defined by specific DNA elements and chromatin features, whereas in metazoans, origin specification is largely sequence-independent and influenced by epigenetic and three-dimensional genome architecture. This review summarises current knowledge of how replication timing is established, regulated, and functionally integrated with chromatin states across eukaryotes, with emphasis on chromatin accessibility, histone modifications and variants, transcriptional regulators, and higher-order genome topology. It also highlights recent genome-wide approaches that map origin licensing, usage, and nascent DNA synthesis at high resolution, revealing dynamic connections between replication, transcription, and nuclear compartmentalisation. Finally, we discuss how disrupted replication timing contributes to replication stress, genome instability, ageing, and cancer. By integrating findings from diverse eukaryotic systems, this provides an updated framework for understanding replication timing as a key layer of genome regulation.