Background and objective Accurate identification of parathyroid adenoma (PA) is essential for minimally invasive treatment of primary hyperparathyroidism (PHPT). The aim of this study is to evaluate the results of parathyroid hormone (PTH) assay in aspirates of suspicious neck lesions and to clarify its reliability in determining whether the lesion is of parathyroid origin. Methods A total of 134 lesions (104 imaging-suspected PA and 30 concomitant thyroid nodules as a control group) of 101 patients were retrospectively analyzed. Patients with positive, negative, or never scintigraphy were included in our study. Ultrasound (US) was performed again and US-guided tissue fine needle aspiration with PTH washout (PTH-WO) was performed from suspicious lesions. A PTH-WO level higher than the patient's serum PTH levels is proposed for a positive test, and a PTH-WO level lower than the upper limit of the laboratory PTH level is proposed for a negative test. A definitive diagnosis was made postoperatively histopathologically. Results PTH-WO levels (median (IQR)) were significantly higher in the positive group (n = 93, 5000 (1600) ng/L) compared to the negative group (n = 11, 17 (13.1) ng/L) and thyroid nodule aspirate group (n = 30, 14 (4.3) ng/L) (p < 0.001). Among 93 PTH-WO-positive lesions, 42 lesions (45.1%) were not identified by parathyroid scintigraphy (PS), 20 (21.5%) lesions were suspiciously PS, and 33 lesions (35.4%) were negative or suspicious by first US findings. Of the 93 patients localized with PTH-WO, two were cystic PAs ablated by aspiration. The final pathology result of 91 operated patients proved PA. The sensitivity, specificity, negative predictive value, and positive predictive value of the PTH-WO were observed as 1.00, 0.82, 0.09, and 0.91, respectively. Conclusion Comprehensive repeated US and PTH-WO from the lesion increase the accurate localization of PAs. PTH-WO is a highly reliable method for differentiating parathyroid lesions from other cervical lesions.