Tumour hypoxia is a negative prognostic and predictive factor owing to multiple contributions to disease progression and resistance to therapy. Several chemical classes of antineoplastic agents, known as bioreductive prodrugs, have been rationally developed to selectively deliver cytotoxic drugs to hypoxic tumour cells. A critical component of translating bioreductive prodrugs to the clinic is the discovery of companion biomarkers that predict sensitivity of tumours to these agents. In this study, we show that defects in homologous recombination repair (HRR) constitute a major pharmacophore-dependent determinant of sensitivity to bioreductive prodrugs. Genetic deletion of the RAD51 paralog Rad51d significantly increased sensitivity of CHO cells to prodrugs of DNA alkylating agents – TH-302 and PR-104A – by a factor comparable to the nitrogen mustard chlorambucil, but only modestly affected the antiproliferative activity of the triazine di-N-oxides SN30000 and tirapazamine. In cell models of triple-negative breast cancer, short hairpin RNA-mediated knockdown of HRR pathway genes BRCA1 and PALB2 similarly increased antiproliferative activity of TH-302, PR-104A and cisplatin. Homozygous knockout of BRCA2 fully inhibited radiation-induced RAD51 focus formation (a measure of HRR) in DLD-1 cells and sensitised them to TH-302, PR-104A and cisplatin but only modestly to SN30000 in vitro. BRCA2 knockout also markedly increased clonogenic cell killing by TH-302 and PR-104A in DLD-1 xenografts, both as monotherapy and in combination with ionising radiation, while SN30000 showed less dependence on BRCA2. These findings suggest that TH-302 and PR-104 may have significant therapeutic activity in cancers that exhibit HRR deficiency. Clinical assays that measure HRR function in tumours should be investigated as predictive biomarkers for stratification of cancer patients likely to respond to these treatments.