Background/Objectives: Since 2021, indeterminate thyroid nodules have been routinely assessed using molecular testing through Quebec’s provincial ThyroSeq v3 pilot project. The primary objective of our study was to compare the molecular and clinicopathological characteristics of Bethesda III and Bethesda IV thyroid nodules evaluated through the pilot project. The secondary objective was to assess the program’s economic impact. Methods: This retrospective study included 934 patients from two McGill University teaching hospitals in Quebec, comprising 593 Bethesda III and 341 Bethesda IV nodules that underwent molecular testing between 2021 and 2025. Eligible nodules were TI-RADS 3 or 4, measured 1–4 cm, and Bethesda III nodules required two consecutive cytology results before testing. Clinicopathological characteristics, histopathological diagnoses, molecular findings, and mutational profiles were analyzed. A projected 10-year cost analysis was also performed. Results: Of the 934 nodules tested, 280 demonstrated a genetic alteration. The risk of malignancy among surgically resected ThyroSeq v3-positive Bethesda III and IV nodules was 68% and 78%, respectively. Bethesda IV nodules demonstrated significantly greater vascular invasion (p = 0.029) and prevalence of oncocytic carcinomas (p = 0.010). Molecular testing was associated with an estimated CAD 1.49 million reduction in healthcare costs over 10 years. Conclusions: Although Bethesda IV nodules exhibited more oncocytic carcinomas and invasion, molecular mutation class distribution and the prevalence of aggressive pathological features were similar between Bethesda categories. Together with the demonstrated economic savings, these findings support continued implementation of publicly funded ThyroSeq v3 testing to improve management of indeterminate thyroid nodules while reducing unnecessary surgery.

Background/Objectives: Since 2021, indeterminate thyroid nodules have been routinely assessed using molecular testing through Quebec’s provincial ThyroSeq v3 pilot project. The primary objective of our study was to compare the molecular and clinicopathological characteristics of Bethesda III and Bethesda IV thyroid nodules evaluated through the pilot project. The secondary objective was to assess the program’s economic impact. Methods: This retrospective study included 934 patients (mean age 55.7 ± 14.3 years; 79.3% female; mean nodule size 2.2 ± 0.8 cm) from two McGill University teaching hospitals in Quebec, comprising 593 Bethesda III and 341 Bethesda IV nodules that underwent molecular testing between 2021 and 2025. Eligible nodules were TI-RADS 3 or 4, measured 1–4 cm, and Bethesda III nodules required two consecutive cytology results before testing. Clinicopathological characteristics, histopathological diagnoses, molecular findings, and mutational profiles were analyzed. A projected 10-year cost analysis was also performed. Results: Of the 934 nodules tested, 280 demonstrated a genetic alteration. The risk of malignancy among surgically resected ThyroSeq v3-positive Bethesda III and IV nodules was 68% and 78%, respectively. Compared with Bethesda III nodules, Bethesda IV nodules demonstrated significantly greater vascular invasion (p = 0.029) and prevalence of oncocytic carcinomas (p = 0.010). Molecular testing was associated with an estimated reduction of 1.49 million Canadian dollars in healthcare costs over 10 years. Conclusions: Although Bethesda IV nodules exhibited more oncocytic carcinomas and invasion, molecular mutation class distribution and the prevalence of aggressive pathological features were similar between Bethesda categories. Together with the demonstrated economic savings, these findings support continued implementation of publicly funded ThyroSeq v3 testing to improve management of indeterminate thyroid nodules while reducing unnecessary surgery.