Updated project metadata. Replication stress (RS) can lead to the formation of DNA double strand breaks (DSBs) and threatens genomic stability. Homologous recombination (HR) pathway is essential to prevent RS and repairs associated DSBs. Upon excessive RS or HR deficiency, DSBs can persist in mitosis, wherein DNA damage response and DSB repair are inhibited. Thus, the fate of DSBs remaining or arising in mitosis remains poorly understood. Here we unwind two distinct roles of the polymerase theta (Polθ) in the maintenance of genomic stability. First, by mass spectrometry and foci screening we show that, in interphase, Polθ interacts with HR proteins and its recruitment to IR-induced DSBs is highly dependent of HR pathway. Secondly, we identify a novel role of the Polθ in counteracting the deleterious effect of DSBs in mitosis. Contrariwise to its interphase recruitment, Polθ recruitment to mitotic DSBs is HR-independent and triggered by mitotic kinases activities. In response to RS or HR deficiency, Polθ localizes to DSBs throughout mitosis and forms nuclear bodies in the G1 daughter cells. In addition, Polθ localizes to centromeres and acentric fragments and activates the mitotic checkpoint. Conversely, Polθ deficiency leads to premature anaphase onset, resulting in an accumulation of mitotic abnormalities. Strikingly, the specific inhibition of Polθ in mitosis kills HR-deficient cells, identifying mitotic Polθ function as its key role in maintaining genome integrity. Our results pinpoint to the mitotic Polθ-mediated replication stress response pathway as a unique vulnerability of cancer cells, with great potential for further investigation.