Introduction. Primary hyperparathyroidism (PHPT) is a condition characterized by disorders of calcium–phosphate metabolism and bone metabolism caused by pathological overproduction of parathyroid hormone (PTH). The diagnosis of overt PHPT is based on the presence of clinical symptoms and laboratory abnormalities typical of this condition: hypercalcemia, hypercalciuria and elevated iPTH levels. Imaging studies are not used for diagnostic purposes; they are performed to localize the parathyroid glands prior to potential surgical treatment. Technetium 99 m sestamibi scintigraphy (Tc99 m-MIBI) is the gold standard in the assessment of pathologically altered parathyroid glands. Other diagnostic options include cervical ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET). Parathyroid biopsy (P-FNAB) with iPTH washout concentration (iPTH-WC) assessment is still an underestimated method of preoperative parathyroid gland localization. Few studies have reported the utility of US-guided P-FNAB in preoperative assessment of parathyroid lesions. The aim of the study was to present our experience with 143 P-FNAB with iPTH-WC assessment. Material and methods. Laboratory results, US findings, P-FNAB complications and comparison with other imaging techniques were described and analyzed. Results. In 133 (93.0) patients, iPTH washout-to-serum ratio exceeded threshold level 0.5 and were classified as positive results. Median iPTH-WC in this group was 16,856 pg/mL, and the iPTH-WC to serum iPTH ratio was 158. There was no correlation between iPTH-WC and serum PTH, serum calcium, parathyroid gland volume and shape index. In the group of 46 operated patients, 44 demonstrated positive iPTH-WC results, which corresponds to a sensitivity of 95.6%. In Tc99-MIBI, radiotracer retention was found in 17 cases (in 24 MIBI performed), which corresponds to a sensitivity of 52.2%. P-FNAB did not cause any major side effects −92.5% of all patients had no or mild adverse events after this procedure. Conclusions. P-FNAB with iPTH-WC is a reliable method in parathyroid adenoma localization during PHPT. Its sensitivity for diagnosis of PHPT is much higher than that of Tc99-MIBI, and in some situations, P-FNAB with iPTH-WC may even replace that method. Furthermore, cost-effectiveness of iPTH-WC is at least similar to that of Tc99-MIBI. Complications of P-FNAB are mild and we can describe this method as a safe procedure.