Purpose: This study aimed to clarify the frequency, phenotypes and molecular spectrum of DUOX2, TPO, and TSHR mutations in patients with congenital hypothyroidism (CH) with enlarged or normal-sized eutopic thyroid glands. Methods: The study cohort included 43 infants and children from 41 unrelated families who had transient or permanent CH with eutopic thyroid glands. Mutation analyses of DUOX2, TPO, and TSHR were performed. In case of extremely low serum thyroglobulin levels, TG analysis was carried out. The functional capacities of novel missense variants of DUOX2 were verified by measuring H2O2 generation in vitro. Results: Of the 43 subjects, 28 (65%) had sequence variants in at least one gene. Twelve different DUOX2 sequence variants, including 7 novel variants were identified in 20 subjects. Sequence variants in TSHR and TPO were identified in 10 and 3 subjects. Of the 20 subjects with DUOX2 variants, 1 subject had transient CH, 9 had mild thyroid dysfunction, and 10 were found to have permanent CH at age 3. A functional analysis of the DUOX2 variants revealed that most variants, other than p.G206V and p.H678R, caused a significant reduction in H2O2 generation. Six of the 43 subjects (14%) had sequence variants in two different genes, suggesting oligogenic causes of CH with eutopic thyroid glands. Conclusions: DUOX2 variants are a relatively common cause of CH with normal-sized or enlarged eutopic thyroid glands. Variable phenotypes were associated with partial loss of functional activity of DUOX2 variants. Oligogenic causes were prominent feature in the development of dyshormonogenic CH.