Hyperparathyroidism is a common endocrine disorder. The precise localization of causal parathyroid gland is crucial to guide surgical treatment. Several studies report the added value of 18F-fluorocholine (FCH) positron emission tomography-computed tomography (PET/CT) as second line imaging but rely on suboptimal first-line imaging using 99mTc-sestaMIBI dual phase scintigraphy. The aim of this study is to evaluate the percentage of successful parathyroid localization with FCH PET/CT after failure of a more sensitive first-line detection protocol associating neck ultrasonography and 99mTc-Pertechnetate/99mTc-sestaMIBI dual tracer subtraction scintigraphy.We included retrospectively 47 patients who underwent a FCH PET/CT as second line imaging for biologically proven primary hyperparathyroidism from November 2016 to October 2018 in Godinot Institute (Reims, France). 99mTc-Pertechnetate/99mTc-sestaMIBI dual tracer subtraction scintigraphy and neck ultrasonography were used as first-line imaging and failed to localize the causal parathyroid lesion in all cases.FCH PET/CT demonstrated at least 1 parathyroid target lesion in 29 patients (62%). 21/29 patients underwent surgery. Target lesions corresponded histologically to hyperfunctioning parathyroid glands for all 21 patients and surgery was followed by hyperparathyroidism biological resolution. Calcium serum levels were associated to FCH PET/CT positivity (P = .002) and a trend toward significance was seen for Parathyroid hormone (PTH) levels (P = .09).FCH PET/CT is a promising tool in second-line parathyroid imaging. Large prospective studies and cost-effectiveness analyses are needed to precise its role.