Abstract: Objectives: To identify the spectrum and prevalence of thyroid dyshormonogenesis causative gene mutations in congenital hypothyroidism (CH) patients. Methods: Blood samples of 89 CH patients were collected, and genomic DNA was extracted. A customized targeted next-generation sequencing panel containing five thyroid dyshormonogenesis -causing genes was designed to detect mutations in the coding regions and exon-intron boundaries of these genes: DUOX2 , TPO, TG, TPO and SLC5A5 . Results: A total of 89 mutations were identified in 61%(54/89) of patients，including DUOX2 (n=41), TG(n=40)and TPO(n=9), no mutation of IYD or SLC5A5 was detected. Among 54 mutations detected patients, 23 patients were two or more mutations detected, and mutations of 11 patients related to two or three genes. Conclusions: Mutations of DUOX2 and TG were frequent detected in Chinese CH patients, while the prevalence of TPO gene mutations was low, IYD and SLC5A5 gene mutation was rare. Multiple site mutations in a single gene or mutations in multiple gene accounts for a part of genetic pathogenesis in CH patients.