Hyperpolarised [1-C]pyruvate MRI (HP-C-MRI) is an emerging metabolic imaging technique that has shown promise for evaluating prostate cancer (PCa) aggressiveness. Accurate tumour delineation on HP-C-MRI is vital for quantitative assessment of the underlying tissue metabolism. However, there is no consensus on the optimum method for segmenting HP-C-MRI, and whole-mount pathology (WMP) as the histopathological gold-standard is only available for surgical patients. Although proton MRI can be used for tumour delineation, this approach significantly underestimates tumour volume, and metabolic tumour segmentation based on HP-C-MRI could provide an important functional metric of tumour volume. In this study, we quantified metabolism using HP-C-MRI and segmentation approaches based on WMP maps, H-MRI-derived T-weighted imaging (T2WI), and HP-C-MRI-derived total carbon signal-to-noise ratio maps (TC-SNR) with an SNR threshold of 5.0. C-labelled pyruvate SNR, lactate SNR, TC-SNR, and the pyruvate-to-lactate exchange rate constant (k) were significantly higher when measured using the TC-SNR-guided approach, which also corresponded to a significantly higher tumour epithelial expression on RNAscope imaging of the enzyme catalysing pyruvate-to-lactate metabolism (lactate dehydrogenase (LDH)). However, linear regression and Bland-Altman analyses demonstrated a strong linear relationship between all three segmentation approaches, which correlated significantly with RNA-scope-derived epithelial LDH expression. These results suggest that standard-of-care T2WI and TC-SNR maps could be used as clinical reference tools for segmenting localised PCa on HP-C-MRI in the absence of the WMP gold standard. The TC-SNR-guided approach could be used clinically to target biopsies towards highly glycolytic tumour areas and therefore to sample aggressive disease with higher precision. KEY POINTS: • T2WI- and TC-SNR-guided segmentations can be used in all PCa patients and do not explicitly require WMP maps. • Agreement between the three segmentation approaches is biologically validated by their strong relationship with epithelial LDH mRNA expression. • The TC-SNR-guided approach can potentially be used to identify occult disease on H-MRI and target the most glycolytically active regions.