Objective: Preoperative location of hyperfunctioning parathyroid glands (HPGs) is vital when planning minimally invasive surgery in patients with primary hyperparathyroidism (PHPT). Dual-isotope subtraction scintigraphy with ^99mTc-MIBI/¹²³Iodide using SPECT/CT and planar pinhole imaging (Di-SPECT) has shown high sensitivity, but is challenged by high radiation dose, time consumption and cost. ¹¹C-Choline PET/CT (faster with a lower radiation dose) is non-inferior to Di-SPECT. We aim to clarify to what extent the two are interchangeable and how often there are discrepancies. Design: This is a prospective, GCP-controlled cohort study. Patients and Measurements: One hundred patients diagnosed with PHPT were included and underwent both imaging modalities before parathyroidectomy. Clinical implications of differences between imaging findings and negative imaging results were assessed. Surgical findings confirmed by biochemistry and pathology served as reference standard. Results: Among the 90 patients cured by parathyroidectomy, sensitivity was 82% (95% confidence interval [CI]: 74%–88%) and 87% (95% CI: 79%–92%) for Choline PET and Di-SPECT, respectively, p =.88. In seven cases at least one imaging modality found no HPG. Of these, neither modality found any true HPGs and only two were cured by surgery. When a positive finding in one modality was incorrect, the alternative modality was correct in approximately half of the cases. Conclusion: Choline PET and Di-SPECT performed equally well and are both appropriate as first-line imaging modalities for preoperative imaging of PHPT. When the first-line modality fails to locate an HPG, additional preoperative imaging with the alternate modality offers no benefit. However, if parathyroidectomy is unsuccessful, additional imaging with the alternate modality has merit before repeat surgery.