Aims:To determine the merit of repeating 50-g glucose challenge test (50-g GCT) at 24-28 weeks of gestation in high-risk pregnant women and the prevalence of early-and late-onset gestational diabetes mellitus (GDM). Methods: This study was conducted in 1447 pregnant women, 344 of them were at risks for GDM undergoing screening by two-step method at first visit. Data were not completely collected in 47 women leaving only 297 for analyses. Fifty-six of 297 pregnant women were diagnosed as early-onset GDM (18.8%) and the other 241 pregnant women were consequently screened at 24-28 weeks of gestation to identify late-onset GDM. Screening data were retrieved, including risks, laboratory test results, baseline characteristics and diagnostic outcomes. Proportion of abnormal second 50-g GCT and the prevalence of late-onset GDM were estimated. Results: The prevalence of late-onset GDM was 17.8%. Among groups of normal 50-g GCT, abnormal 50-g GCT and abnormal 1 oral glucose tolerance test (OGTT) value at first test, the proportions of abnormal 50-g GCT at second test were 38.3%, 59.5% and 79.3%, and the prevalence of late-onset GDM among each group were 18.8%, 8.3% and 41.4%, consecutively. This lead to prevalence of GDM in this high-risk group of 33.3% and overall among 1447 women was 7.9%. Conclusion: There were high proportions of abnormal 50-g GCT at second test in the groups with abnormal 50-g GCT and abnormal 1 OGTT value at first test (59.5% and 79.3%). Authors suggested doing OGTT without repeating 50-g GCT in these groups coming at 24-28 weeks of gestation.