Predictive biomarkers are urgently needed for individualization of radiation therapy and treatment with radiosensitizing anticancer agents. Genomic profiling of human cancers provides us with unprecedented insight into the mutational landscape of genes directly or indirectly involved in the response to radiation-induced DNA damage. However, to what extent this wealth of structural information about the cancer genome produces biomarkers of sensitivity to radiation remains to be seen. Investigators are increasingly studying the subnuclear accumulation (ie, foci) of proteins in the DNA damage response (DDR), such as gamma-H2AX, 53BP1, or RAD51, as a surrogate of treatment sensitivity. Recent findings from preclinical studies have demonstrated the predictive potential of DDR foci by correlating foci with clinically relevant end points such as tumor control probability. Therefore, preclinical investigations of DDR foci responses are increasingly moving into cells and tissues from patients, which is the major focus of this review. The advantage of using DDR foci as functional biomarkers is that they can detect alterations in DNA repair due to various mechanisms. Moreover, they provide a global measurement of DDR network function without needing to know the identities of all the components, many of which remain unknown. Foci assays are thus expected to yield functional insight that may complement or supersede genomic information, thereby giving radiation oncologists unique opportunities to individualize cancer treatments in the near future.