The cellular response to DNA double strand breaks (DSBs) involves the ordered assembly of repair proteins at or near sites of damage. This process is mediated through post-translational protein modifications that include both phosphorylation and ubiquitylation. Recent data have demonstrated that recruitment of the repair proteins BRCA1, 53BP1, and RAD18 to ionizing irradiation (IR) induced DSBs is dependent on formation of non-canonical K63-linked polyubiquitin chains by the RNF8 and RNF168 ubiquitin ligases. Here we report a novel role for K63-ubiquitylation in response to replication-associated DSBs that contributes to both cell survival and maintenance of genome stability. Suppression of K63-ubiquitylation markedly increases large-scale mutations and chromosomal aberrations in response to endogenous or exogenous replication-associated DSBs. These effects are associated with an S-phase specific defect in DNA repair as revealed by an increase in residual 53BP1 foci. Use of both knockdown and knockout cell lines indicates that unlike the case for IR-induced DSBs, the requirement for K63-ubiquitylation for the repair of replication associated DSBs was found to be RNF8-independent. Our findings reveal the existence of a novel K63-ubiquitylation dependent repair pathway that contributes to the maintenance of genome integrity in response to replication-associated DSBs.