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Perturbation of DNA replication, for instance by hydroxyurea-dependent dNTP exhaustion, often leads to stalling or collapse of replication forks. This triggers a replication stress response that stabilizes these forks, activates cell cycle checkpoints, and induces expression of DNA damage response genes. While several factors are known to act in this response, the full repertoire of proteins involved remains largely elusive. Here, we develop Replication-IDentifier (Repli-ID), which allows for genome-wide identification of regulators of DNA replication in Saccharomyces cerevisiae. During Repli-ID, the replicative polymerase epsilon (Pol ε) is tracked at a barcoded origin of replication by chromatin immunoprecipitation (ChIP) coupled to next-generation sequencing of the barcode in thousands of hydroxyurea-treated yeast mutants. Using this approach, 423 genes that promote Pol ε binding at replication forks were uncovered, including LGE1 and ROX1. Mechanistically, we show that Lge1 affects replication initiation and/or fork stability by promoting Bre1-dependent H2B mono-ubiquitylation. Rox1 affects replication fork progression by regulating S-phase entry and checkpoint activation, hinging on cellular ceramide levels via transcriptional repression of SUR2. Thus, Repli-ID provides a unique resource for the identification and further characterization of factors and pathways involved in the cellular response to DNA replication perturbation.