The study investigates the role of unprocessed genomic uracil in DNA replication stress (RS) in cancer cells. Genomic uracil, a common base alteration, is shown to induce RS without being processed by base excision repair (BER). In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates, leading to slowed DNA replication forks and enhanced PrimPol-mediated repriming, resulting in single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells with high levels of genomic uracil upregulate UNG2 to limit RS and are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These findings reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability in cancer cells.The study investigates the role of unprocessed genomic uracil in DNA replication stress (RS) in cancer cells. Genomic uracil, a common base alteration, is shown to induce RS without being processed by base excision repair (BER). In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates, leading to slowed DNA replication forks and enhanced PrimPol-mediated repriming, resulting in single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells with high levels of genomic uracil upregulate UNG2 to limit RS and are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These findings reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability in cancer cells.