ObjectiveNear‐infrared autofluorescence (NIRAF) characteristics of parathyroid glands in primary hyperparathyroidism (pHPT) vary, with unclarity regarding the underlying mechanism. Similarly, 99mTc‐sestamibi uptake in diseased parathyroid glands is variable. There is a suggestion that oxyphilic cell content may influence both imaging modalities. This study aims to analyze the relationship between NIRAF imaging characteristics, 99mTc‐sestamibi uptake, and cellular composition in pHPT.Study DesignRetrospective analysis of an Institutional Review Board‐monitored prospective database.SettingSingle tertiary referral center.MethodsNIRAF characteristics of parathyroid glands of patients with pHPT between 2019 and 2024 were compared with 99mTc‐sestamibi scan findings from a prospective database. Using third‐party software, brightness intensity and heterogeneity index (HI) of the glands were calculated. A subgroup of parathyroid glands obtained from consecutive patients with pHPT in 2020 to 2021 underwent histological analysis.ResultsA total of 428 patients with 638 diseased parathyroid glands were analyzed. Forty‐seven percent of the glands showed an uptake on 99mTc‐sestamibi scans. The brightness intensity of the NIRAF signals from parathyroid glands that were seen versus not seen on sestamibi was 2.1 versus 2.3 (P = .002) and HI 0.18 versus 0.17 (P = .35), respectively. On multivariate analysis, low autofluorescence intensity, high gland volume, and single adenoma were associated with detectability on 99mTc‐sestamibi scan (P < .0001). Intraglandular adipose tissue content was lower in diseased glands that were detected on 99mTc‐sestamibi scans (0% vs 5%, P < .0001).ConclusionOur findings indicate an inverse relationship between autofluorescence intensity and detectability on 99mTc‐sestamibi scans and a lack of correlation between different cell types and autofluorescence properties.