Men with high-risk prostate cancer (PCa) are treated with androgen deprivation therapy (ADT) and radiotherapy, but the disease reoccurs in 30% of patients. Biochemical recurrence of PCa after treatment is influenced by tumour hypoxia. Tumours with high levels of hypoxia are aggressive, resistant to treatment, and have increased metastatic capacity. Gene expression signatures derived from diagnostic biopsies can predict tumour hypoxia and radiosensitivity, but none are in routine clinical use, due to concerns about the applicability of these biomarkers to new patient cohorts. There has been no or limited testing in cohorts of high-risk PCa. We generated transcriptomic data for cohorts of high-risk PCa patients. Patients were treated with ADT followed by external beam radiotherapy with or without a brachytherapy boost. Biomarkers curated from the literature were calculated from pre-treatment biopsy gene expression data. The primary endpoint for survival analyses was biochemical recurrence-free survival (bRFS) and the secondary endpoints were distant metastasis-free survival (DMFS) and overall survival. The performance of the selected biomarkers was poor, with none achieving prognostic significance for bRFS or DMFS in any cohort. The brachytherapy boost cohort received shorter durations of ADT than the conventionally fractionated or hypofractionated cohorts (Wilcoxon rank sum test, p=2.1 × 10 and 2.3 × 10 respectively) and had increased risk of distant metastasis (log-rank test, p=8 × 10 ). There were no consistent relationships between biomarker score and outcome for any of the endpoints. Hypoxia and radiosensitivity biomarkers were not prognostic in high-risk PCa patients treated with ADT plus radiotherapy. We speculate that the lack of prognostic capability could be caused by the variable hypoxia-modifying effects of the ADT that these high-risk patients received before and during definitive treatment with radiotherapy. A deeper understanding of biomarker construction, performance and inter-cohort transferability in relation to patient characteristics, sample handling and treatment modalities is required before hypoxia biomarkers can be recommended for routine clinical use in the pre-treatment setting. [Abstract copyright: Copyright © 2024. Published by Elsevier Inc.]