Aims <p>To compare the effect on maternal and neonatal outcomes of 75-g oral glucose tolerance test (75-g OGTT) versus 50-g glucose challenge test (GCT) plus 75-g OGTT for diagnosis of gestational diabetes mellitus (GDM).</p> Materials and methods <p>A non-randomized trial was conducted in a tertiary hospital between January and December 2020. Participants were assigned into the one-step (i.e., 75-g OGTT) and two-step screening (50-g GCT plus 75-g OGTT) groups. Primary outcomes were GDM, hypertensive disorder in pregnancy (HDP), macrosomia, and Cesarean section.</p> Results <p>2265 eligible participants were enrolled in the trial, including 1130 in the one-step group and 1135 in the two-step group. GDM was diagnosed in 197 (17.4%) participants in the one-step group and 123 (10.8%) in the two-step group (OR, 1.94; 95% CI, 1.49, 2.54). There was only borderline statistical significance in the difference of HDP between two groups (OR, 0.64; 95% CI, 0.40, 1.01), while all other outcomes showed no statistically significant differences. In pregnant women with high risk factors for GDM (maternal age ≥ 35 years, pre-pregnancy BMI ≥ 24 kg/m², multipara, history of GDM, or family history of diabetes), the incidence of GDM was higher (OR, 1.74; 95% CI, 1.19, 2.56) for the one-step versus two step-step screening, while the incidences of HDP (OR, 0.51; 95% CI, 0.29, 0.90) and macrosomia (OR, 0.62; 95% CI, 0.39, 0.97) were lower.</p> Conclusions <p>The one-step screening at least performs as well as the two-step screening, potentially more suitable for Chinese pregnant women with high risk factors for GDM.</p> <p>The study was registered at the Chinese Clinical Trial Registry (ChiCTR2100054505) on Dec 18^th, 2021.</p>