Primary hyperparathyroidism (HPT) in 80% of patients is due to a solitary parathyroid adenoma, while in 20% multigland pathology exists, usually hyperplasia [Scott-Coombes, Surgery, 21(12):309–312, 2003]. Despite recent advances in minimally invasive parathyroidectomy, better preoperative localisation techniques and intraoperative parathyroid hormone (PTH) monitoring, a 4% failure rate [Grant CS, Thompson G, Farley D, Arch Surg, 140:47–479, 2005] persists making accurate differentiation between adenomas and hyperplasia of prime importance. We investigated the ability of Raman spectroscopy to accurately differentiate between parathyroid adenomas and hyperplasia. Raman spectra were measured at defined points on the parathyroid tissue sections using a bench-top microscopy system. Multivariate analysis of the spectra was carried out to construct a diagnostic algorithm correlating spectral results with the histopathological diagnosis. A total of 698 spectra were analysed. Principal-component (PCA)-fed linear discriminant analysis (LDA) used to construct a diagnostic algorithm. Detection sensitivity for parathyroid adenomas was 95% and hyperplasia was 93%. These preliminary results indicate that Raman spectroscopy is potentially an excellent tool to differentiate between parathyroid adenomas and hyperplasia.