Background and objectives The accurate identification of hyper functioning parathyroid gland is needed for definitive surgical treatment in primary hyperparathyroidism. Ultrasonography and 99mTechnetium sestamibi scintigraphy are the two most used methods with varying sensitivities. This study aimed to assess the value of parathyroid hormone (PTH) assay in preoperative ultrasound guided fine needle aspiration (FNA)-PTH washout fluid to verify the correct localisation of lesions with negative or inconclusive scintigraphy results. Methods We evaluated data of 28 lesions in 21 patients who underwent US-guided parathyroid fine-needle aspiration (FNA) with PTH washout, retrospectively. The PTH washout results and the reports of parathyroid surgery and imaging studies were reviewed. Results Of operated 28 lesions 23 had positive and 5 had negative washout results. The median FNA-PTH washout was 2315.5 pg/ ml (min-max: 12.3-6978 pg/ ml). The calculated sensitivity of FNA-PTH washout was 85.7% and the specifity was 28.6%. The positive and negative predictive values were 78.3% and 40.0%, respectively. Conclusions FNA-PTH can be used to establish the nature of the lesion, discriminate parathyroid gland from thyroid lesions or cervical lymph nodes, improving the surgical outcomes. It can be used to localise parathyroid lesions preoperatively when negative or discordant ultrasound and scintigraphy findings are obtained.