Our previous study showed that paternal subclinical hypothyroidism (SCH) had a detrimental effect on the clinical outcomes of assisted reproductive technologies. However, it remains to be determined whether paternal SCH affects sperm DNA integrity.To investigate the association between SCH and sperm DNA fragmentation in men seeking infertility care.This cross-sectional study included 4,983 men with euthyroidism and 418 men with SCH seeking infertility treatment in a tertiary care academic medical center between January 2017 and December 2021. The outcome measures were the absolute DNA fragmentation index (DFI) and the risk of abnormal DFI (defined as a DFI of ≥25% or ≥30%).The mean (standard deviation) age of men with euthyroidism and men with SCH was 34.20 (5.97) and 35.35 (6.48) years, respectively (p < 0.001). The difference in DFI was not statistically significant [adjusted mean: 19.7% vs. 18.9% in the SCH and euthyroidism groups, respectively, p = 0.07] after confounder adjustment. A DFI of ≥25% was significantly more frequent in men with SCH (20.57%) than in men with euthyroidism (14.49%) after confounder adjustment [odds ratio (OR) (95% CI): 1.43 (1.09-1.88)]. A DFI of ≥30% was also significantly more common in men with SCH (11.72%) than in men with euthyroidism (6.74%) [OR (95%CI): 1.84 (1.34-2.52)]. In addition, thyroid-stimulating hormone (TSH) concentration was significantly associated with an increased risk of having a DFI of ≥25% (p < 0.001) or ≥30% (p = 0.011).SCH was significantly associated with an increased risk of an abnormal DFI.© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.