BackgroundNewborn screening (NBS) is an important and successful public health program that helps improve the long-term clinical outcomes of newborns by providing early diagnosis and treatment of certain inborn diseases. The development of next-generation sequencing (NGS) technology provides new opportunities to expand current newborn screening methodologies.MethodsWe designed a a newborn genetic screening (NBGS) panel targeting 135 genes associated with 75 inborn disorders by multiplex PCR combined with NGS. With this panel, a large-scale, multicenter, prospective multidisease analysis was conducted on dried blood spot (DBS) profiles from 21,442 neonates nationwide.ResultsWe presented the positive detection rate and carrier frequency of diseases and related variants in different regions; and 168 (0.78%) positive cases were detected. Glucose-6-Phosphate Dehydrogenase deficiency (G6PDD) and phenylketonuria (PKU) had higher prevalence rates, which were significantly different in different regions. The positive detection ofG6PDvariants was quite common in south China, whereasPAHvariants were most commonly identified in north China. In addition, NBGS identified 3 cases with DUOX2 variants and one with SLC25A13 variants, which were normal in conventional NBS, but were confirmed later as abnormal in repeated biochemical testing after recall. Eighty percent of high-frequency gene carriers and 60% of high-frequency variant carriers had obvious regional differences. On the premise that there was no significant difference in birth weight and gestational age, the biochemical indicators ofSLC22A5c.1400C > G andACADSBc.1165A > G carriers were significantly different from those of non-carriers.ConclusionsWe demonstrated that NBGS is an effective strategy to identify neonates affected with treatable diseases as a supplement to current NBS methods. Our data also showed that the prevalence of diseases has significant regional characteristics, which provides a theoretical basis for screening diseases in different regions.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12519-022-00670-x.