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Background: Frozen section is the standard method to histologically distinguish parathyroid tissue from thyroid tissue during endocrine neck surgery. Frozen section can be time-consuming and costly. Its drawback is that it is to be performed only after the removal of a suspected pathological tissue. This study demonstrates the use of probe-based confocal laser endomicroscopy (pCLE) to confirm histology prior to tissue resection., Design: A prospective, single-institution, nonrandomized study was conducted. No sample size calculation was performed for this observational trial. The primary objective was the description of histological rendering of normal and pathological tissues through pCLE. Real-time in vivo fluorescence microscopy imaging was performed with the CystoFlex UHD probe after intravenous injection of 2.5 mL of 10% fluorescein sodium., Results: Eleven patients with hyperparathyroidism and thyroid conditions were included. A total of 104 videos showing thyroid, parathyroid, adipose tissue, muscle, laryngeal nerve, and lymph nodes were recorded. Videos were compared with visual information and pathological samples (when sampling was indicated). Thyroid tissue could be identified based on the presence of colloid follicles (intensely fluorescent area surrounded by a small ridge of low-fluorescence epithelial cells) including the pathognomonic aspect of resorption vacuole. Parathyroid tissue could be identified based on a regular, "diamond-shaped" capillary network encompassing parathyroid chief cells. Blinded reinterpretation of pCLE videos demonstrated an 89.3% sensitivity and a 90% specificity as compared with histology in tissue recognition., Conclusion: This pilot study describes representative renderings of intraoperative pCLE to nontraumatically differentiate thyroid, parathyroid, and lymph nodes before surgical removal.