It is evident that for a significant proportion of prostate cancer patients radiotherapy provides no survival benefit, instead exposing patients to risk of associated side-effects. Biomarker identification to differentiate radiosensitive and resistant tumour subtypes will ultimately lead to improved treatment response and survival benefit for patients. Both a candidate gene approach and unbiased screen were utilised to search for biomarkers of radiotherapy response in prostate cancer cell lines. NF-κB family members (RelA, RelB, c-Rel, NF-κB1 and NF-κB2) make ideal candidate biomarkers for predicting response due to their integral role in inflammation and apoptosis. Characterisation of NF-κB gene and protein expression profiles, performed using quantitative PCR and immunoblotting, was compared to cell survival, to assess potential contribution to radiotherapy resistance. No significant difference in NF-κB gene expression was apparent between cell lines of differing levels of radioresponsiveness. Certain cell lines demonstrated basal activation of various NF-κB proteins but this also did not correlate with response. However, radioresistant PC-3 cells substantially increased RelB nuclear translocation six hours post-irradiation, not evident in more sensitive cell lines. This indicates this family member may aid cell survival following radiotherapy. RNA-seq analysis of transcriptional variation between radioresistant and sensitive prostate cancer cell lines revealed differential gene expression in DNA repair and replication, apoptosis and cell-cycle progression. Radioresistant PC-3 cells upregulated activation of DNA repair pathways, in contrast, the sensitive cell line, LNCaP, downregulated expression of this pathway. Radioresistance was also associated with a greater number of gene expression changes indicating radiosensitivity and cell death may be a more passive process requiring fewer expression alterations. This suggests post-radiotherapy cell survival is triggered by a tumour’s ability to actively repair DNA damage enabling avoidance of apoptosis. In addition, genes identified in these DNA repair pathways may prove to be valuable predictive biomarkers of response and targets for sensitisation.