The main aim of this study was to investigate the possible association between 18F–choline uptake and histopathological features of prostate biopsies such as the Gleason Group and the expression of both epithelial to mesenchymal transition (vimentin) and bone mineralization (bone morphogenetics protein (BMP)-2, runt-related transcription factor 2 (RUNX2), receptor activator of nuclear factor-κB ligand (RANKL), vitamin D receptor (VDR), and pentraxin 3 (PTX3) in situ biomarkers. To this end, we enrolled 79 consecutive prostate cancer patients that underwent both the 18F–choline PET/CT analysis and the prostate bioptic procedure. The standardized uptake value (SUV) average values were collected from 18F–choline PET/CT analysis whereas Gleason Group and immunostaining data were collected from paraffin-embedded sections. Histological classification showed a heterogenous population including both low/intermediate and high-grade prostate cancers. A significant increase of 18F–choline uptake in high-grade prostate lesions (Gleason Score ≥8) was found. Also, linear regression analysis showed a significant correlation between 18F–choline uptake and the number of vimentin, RANKL, VDR, or PTX3 positive prostate cancer cells. Conversely, we observed no significant association between 18F–choline uptake and the expression of bone biomarkers involved in the early phases of osteoblast differentiation (BMP-2, RUNX2). In conclusion, results here reported can lay the foundation for the use of 18F–choline positron emission tomography (PET)/computed tomography (CT) as a diagnostic tool capable of identifying high-grade prostate cancer lesions expressing bone biomarkers.