Objective To evaluate the prevalence and natural course of autoimmune and non-autoimmune subclinical hypothyroidism (SH) in Down syndrome (DS) children and adolescents. Design Prospective multicenter study. Methods For the study, 101 DS patients with SH (TSH 5–10 mIU/L; FT4 12–22 pmol/L), aged 2–17 years at SH diagnosis were enrolled. Annual monitoring of TSH, FT4, BMI, height, and L-thyroxine dose was recorded for 5 years. Thyroid autoimmunity was tested at diagnosis and at the end of follow-up. Results Thirty-seven out of 101 patients displayed autoantibody positivity (group A); the remaining 64 were classified as non-autoimmune SH (group B). Group A was characterized by higher median age at SH diagnosis and by more frequent family history of thyroid disease (6.6 vs 4.7 years, P = 0.001; 32.4% vs 7.8%, P = 0.001 respectively), whereas congenital heart defects were more common in group B (65.6% vs 43.2%, P = 0.028). Gender, median BMI (SDS), height (SDS), FT4, and TSH were similar in both groups. At the end of follow-up: 35.1% of group A patients developed overt hypothyroidism (OH) vs 17.2% of group B (P = 0.041); 31.25% of group B vs 10.8% of group A became biochemically euthyroid (P = 0.02); and 37.8% of group A vs 51.5% of group B still had SH condition (P = 0.183). Logistic regression suggested autoimmunity (OR = 3.2) and baseline TSH values (OR = 1.13) as predictive factors of the evolution from SH to OH. Conclusions In DS children, non-autoimmune SH showed higher prevalence and earlier onset. The risk of thyroid function deterioration over time seems to be influenced by thyroid autoimmunity and higher baseline TSH values.