The prostate gland is the most common site of pathology in human males. Using the urethra as an anatomical reference point, it can be divided into three distinct zones known as the transition zone (TZ), peripheral zone (PZ) and central zone (CZ). The pathological conditions of benign prostatic hypertrophy and/or prostate adenocarcinoma are highly prevalent in this gland. This preliminary study set out to determine whether biochemical intra-individual differences between normal prostate zones could be identified using Raman spectroscopy with subsequent exploratory analyses. A normal (benign) prostate transverse tissue section perpendicular to the rectal surface and above the verumontanum was obtained in a paraffin-embedded block. A 10-µm-thick slice was floated onto a gold substrate, de-waxed and analysed using Raman spectroscopy (200 epithelial-cell and 140 stromal spectra/zone). Raman spectra were subsequently processed in the 1800–367 cm⁻¹ spectral region employing principal component analysis (PCA) to determine whether wavenumber–intensity relationships expressed as single points in hyperspace might reveal biochemical differences associated with inter-zone pathological susceptibility. Visualisation of PCA scores plots and their corresponding loadings plots highlighted 781 cm⁻¹ (cytosine/uracil) and 787 cm⁻¹ (DNA) as the key discriminating factors segregating PZ from less susceptible TZ and CZ epithelia (P < 0.001). Conversely, 1459 cm⁻¹ (lipids and proteins) and 1003 cm⁻¹ (phenylalanine) were identified as the key biochemical factor distinguishing TZ from CZ epithelia (P < 0.05). All stromal zones were discriminated by the protein/lipid region (1459 cm⁻¹ and 1100 cm⁻¹) with DNA/RNA region (781 cm⁻¹ and 787 cm⁻¹) only highlighted between PZ and CZ (P < 0.05). This novel approach identifies biochemical markers that may have aetiological functional roles towards susceptibility of human prostate zones to specific pathological conditions.