Unprocessed genomic uracil induces DNA replication stress (RS) in cancer cells without being processed by base excision repair (BER). In UNG-deficient cells, genomic uracil accumulates, slowing replication forks and enhancing PrimPol-mediated repriming, which generates single-stranded DNA gaps. ATR inhibition in these cells blocks repair of uracil-induced gaps, increasing replication fork collapse and cell death. Cancer cells with high genomic uracil levels upregulate UNG2 to limit RS, making them hypersensitive to ATR inhibitors and drugs that increase genomic uracil. These findings reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability in cancer cells. Genomic uracil impairs replication forks by causing ssDNA gaps, which are repaired by ATR. ATRi prevents this repair, leading to DNA damage and cell death. High UNG2 expression in cancer cells helps suppress RS, but these cells are sensitive to ATRi and drugs that increase genomic uracil. Combining ATRi with PMX or dUTPase inhibitors enhances ATRi sensitivity in UNG-dependent cancer cells. This study highlights the role of unprocessed genomic uracil in RS and provides a strategy for targeting cancer cells with ATRi and other agents that increase genomic uracil.Unprocessed genomic uracil induces DNA replication stress (RS) in cancer cells without being processed by base excision repair (BER). In UNG-deficient cells, genomic uracil accumulates, slowing replication forks and enhancing PrimPol-mediated repriming, which generates single-stranded DNA gaps. ATR inhibition in these cells blocks repair of uracil-induced gaps, increasing replication fork collapse and cell death. Cancer cells with high genomic uracil levels upregulate UNG2 to limit RS, making them hypersensitive to ATR inhibitors and drugs that increase genomic uracil. These findings reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability in cancer cells. Genomic uracil impairs replication forks by causing ssDNA gaps, which are repaired by ATR. ATRi prevents this repair, leading to DNA damage and cell death. High UNG2 expression in cancer cells helps suppress RS, but these cells are sensitive to ATRi and drugs that increase genomic uracil. Combining ATRi with PMX or dUTPase inhibitors enhances ATRi sensitivity in UNG-dependent cancer cells. This study highlights the role of unprocessed genomic uracil in RS and provides a strategy for targeting cancer cells with ATRi and other agents that increase genomic uracil.