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The relationship between duty hours (DH) and the performance of postgraduate residents is needed to establish appropriate DH limits. This study explores their relationship using the General Medicine In-training Examination (GM-ITE). In this cross-sectional study, GM-ITE examinees of 2019 had participated. We analyzed data from the examination and questionnaire, including DH per week (eight categories). We examined the association between DH and GM-ITE score, using random-intercept linear models with and without adjustments. Five thousand five hundred and ninety-three participants (50.7% PGY-1, 31.6% female, 10.0% university hospitals) were included. Mean GM-ITE scores were lower among residents in Category 2 (45–50 h; mean score difference, −1.05; p < 0.001) and Category 4 (55–60 h; −0.63; p = 0.008) compared with residents in Category 5 (60–65 h; Reference). PGY-2 residents in Categories 2–4 had lower GM-ITE scores compared to those in Category 5. University residents in Category 1 and Category 5 showed a large mean difference (−3.43; p = 0.01). DH <60–65 h per week was independently associated with lower resident performance, but more DH did not improve performance. DH of 60–65 h per week may be the optimal balance for a resident's education and well-being. [ABSTRACT FROM AUTHOR]