Rare diseases are characterized by the extremely low prevalence of individual conditions, and large-scale population-based epidemiological data remain limited. In this study, we aimed to predict the incidence of 42 rare diseases included in the Chinese Rare Disease Catalog based on large-scale newborn targeted genetic screening data. A total of 33,894 newborns from 14 provinces in China were enrolled. Targeted next-generation sequencing of 465 disease-causing genes was performed, and pathogenic/likely pathogenic (P/LP) variants were classified according to the American College of Medical Genetics and Genomics (ACMG)guidelines. Diseases with complete gene coverage were screened from China’s First and Second Lists of Rare Diseases, and predicted incidence was calculated according to different inheritance patterns. Among the listed 207 rare diseases, 82 were covered in this study, of which 42 had complete gene coverage involving 62 pathogenic genes. In 33,894 newborns, 4,657 P/LP variants were detected across 57 genes, with an average of 0.137 variants per individual. Diseases with higher predicted incidence included phenylketonuria (1/5,060), Wilson disease (1/12,200), primary carnitine deficiency (1/17,700), citrullinemia (1/29,600), and glycogen storage disease (types I and II, 1/63,600). This study systematically predicted the incidence of 42 rare diseases with fully covered genes based on large-scale Chinese newborn genomic data. Targeted genetic screening can serve as an effective first-tier combined strategy within the existing newborn screening and provides important evidence-based data for early intervention and public health policies for rare diseases.