Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common X-linked enzymopathies caused by G6PD gene variant. We aimed to provide the characteristics of G6PD deficiency and G6PD gene variant distribution in a large Chinese newborn screening (NBS) population. We investigated the prevalence of G6PD in China from 2013 to 2017. Then we examined G6PD activity and G6PD gene in representative Chinese birth cohort to explore the distribution of G6PD gene variant in 2016. We then performed multicolour melting curve analysis (MMCA) to classify G6PD gene variants in 10,357 neonates with activity-confirmed G6PD deficiency, and DNA Sanger sequencing for G6PD coding exons if hot site variants were not found. The screened population, organizations and provinces of G6PD deficiency were increased from 2013 to 2017 in China. The top 5 frequency of G6PD gene variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, and c.871G>A and varied in different provinces, with regional and ethnic features, and 4 pathogenic variant sites (c.152C>T, c.290A>T, c.697G>C, and c.1285A>G) were firstly reported. G6PD deficiency mainly occurs in South China, and the frequency of G6PD gene variant varies in different regions and ethnicities. This article is protected by copyright. All rights reserved.