© 2015 Elsevier Inc. Background Proper localization is crucial in performing minimally invasive parathyroidectomy for primary hyperparathyroidism. Ultrasonography (US) and Tc-99m sestamibi (MIBI) scintigraphy are common methods used for localization. As the appearance and activity of the thyroid gland may impact parathyroid localization, the purpose of this study was to determine how exogenous use of the thyroid hormone, levothyroxine (LT), affects parathyroid localization. Methods Adult patients with non-familial primary hyperparathyroidism who underwent initial parathyroidectomy from 2000-2014 were retrospectively identified. LT (+LT) and non-LT (-LT) patients were matched 1:3 based on age, gender, goiter status, and preoperative parathyroid hormone levels. Subgroup analysis was performed on patients previously treated with radioactive iodine and patients undergoing single adenoma resection. Results Of the 1737 patients that met inclusion criteria, 286 were on LT at the time of their parathyroid localization scan. Use of LT did not impact the percentage of correct MIBI localization scans when compared with -LT patients (P = 0.83). Interestingly, use of LT significantly hindered localization by US in comparison with the -LT group (48.4 versus 62.2%, P < 0.01). When examining only patients where a single upper gland was removed, the +LT group was less likely to have a correct US compared with the -LT group (50 versus 72.8%, P < 0.01). However, there was no difference in US accuracy for patients who only had a single lower gland removed (P = 0.51). Conclusions Exogenous LT is associated with impaired parathyroid localization with US but not MIBI. Surgeons should be aware of localization efficiency for this subset of patients in the era of personalized medicine and cost effectiveness.