The aim of this prospective pilot study was to evaluate the feasibility of a new hybrid imaging modality, free-hand single-photon computed tomography/ultrasonography (fhSPECT/US), for preoperative localization of parathyroid adenomas and to compare its performance with conventional ultrasonography and SPECT/CT. Twelve patients diagnosed with primary hyperparathyroidism underwent sequentially US and parathyroid scintigraphy, including SPECT/CT, followed by fhSPECT/US, allowing for real-time fusion between US and freehand-generated gamma-camera images. The fhSPECT/US detection rates were correlated with histopathology, when available, or with the imaging modality showing the most lesions. Based on a per patient analysis, the detection rate was significantly different when comparing SPECT/CT to fhSPECT/US (p= 0.03), and not significantly different when comparing SPECT/CT to US (p= 0.16) and US to fhSPECT/US (p= 0.08). Based on a per-lesion analysis, the detection rate of SPECT/CT was significantly higher than that of US (p= 0.01) and fhSEPCT/US (p= 0.003), and there was no significant difference in detection rate when comparing US to fhSPECT/US (p= 0.08). The main perceived limitations of fhSPECT/US in lesion detection were: (i) lesions localized at a depth ≥4.5 cm; (ii) imperfect image fusion due to tissue compression; (iii) limited spatial manipulation ability of the SPECT mobile camera handheld probe; and (iv) a wide spread of detected activity. In conclusion, clinical use of fhSPECT/US for localization of parathyroid adenomas is feasible, but shows lower sensitivity than conventional modalities and requires technical improvements.