Purpose: To determine the frequency of maturity-onset diabetes of the young (MODY) in two selected cohorts of Chinese children with diabetes and clinically suspected MODY, using next-generation sequencing (NGS). Methods: 93 children who met the comprehensive criteria of suspected MODY were enrolled in two cohorts. A custom NGS panel or a whole exon group was used for sequencing. Results: We identified 55/93 (59.1%) children with pathogenic and likely pathogenic MODY variants. 42 (76.3%) were confirmed to have the GCK (MODY2) mutation. Additionally, five had the HNF1A (MODY3), two the HNF1B (MODY5), one the 17q12 microdeletion (MODY5), two the HNF4A (MODY1), two the ABCC8 (MODY12), and one the PDX1 mutation (MODY4). Of these, 13 novel variants were detected in different genes.By comparing the gene-positive with gene-negative children, we found that discriminatory factors for MODY at diagnosis included lower HbA1c (7.4% vs. 10.2% [53 vs. 86 mmol/mol]; P=0.002), lower BMI z score (0.2 vs. 1.0; P=0.01), lower onset age (8.1 vs. 11.2 years; P=0.001), and lower C-peptide (1.4 vs. 2.5 ng/mL; P=0.02). Conclusion: The criteria used in this study for screening MODY are effective, and MODY2 is the most common subtype (76%), followed by MODY3 and MODY5. Some rare MODY subtypes have been reported in Chinese children.