Abstract: Background　21-Hydroxylase deficiency (21-OHD) is an autosomal recessive disorder caused by pathogenic variants in the CYP21A2 gene. The CYP21A2 gene and its highly homologous pseudogene, CYP21A1P, are prone to recombination events, posing significant challenges for carrier screening using next-generation sequencing (NGS). Furthermore, data on large-scale carrier screening for CYP21A2 in the general reproductive-aged population remain limited. Objective　To determine the carrier frequency and variant spectrum of CYP21A2 pathogenic variants in the general reproductive-aged Chinese population, thereby providing essential data to inform population-based screening. Additionally, to evaluate the performance of a novel PCR-based enrichment strategy combined with NGS (PCR-NGS) designed to overcome pseudogene interference and enhance the accuracy and feasibility of screening. Methods　This prospective, multicenter observational study recruited 33 104 asymptomatic cases planning pregnancy or in early pregnancy from 12 tertiary hospitals across China from July 2022 to October 2023. Genomic DNA was extracted from peripheral blood. The CYP21A2 functional gene was specifically enriched via PCR using unique primers, followed by targeted NGS and bioinformatic analysis. High-risk couples and individuals carrying multiple variants identified by NGS were subsequently validated using third-generation sequencing (TGS). Results　Initial NGS screening identified 636 carriers. TGS validation revealed that among individuals flagged with multiple variants, 3 were mild patients with pathogenic variants in trans, and 2 were healthy individuals with true gene duplication. After correction, the final cohort comprised 631 carriers, yielding an overall carrier frequency of 1/52.46. The carrier frequency was 1/53.92 (308/16 610) in females and 1/51.07 (323/16 494) in males. Thirtyfour distinct pathogenic variants were detected, with 88.01% originating from pseudogene-derived microconversions. The three most prevalent variants were c.844G＞T (allele frequency 0.31%), c.293-13C＞G (0.22%), and c.518T＞A (0.15%). Conclusion　This study systematically delineates the CYP21A2 pathogenic variant landscape in the Chinese reproductive-aged population. The established PCR-NGS protocol effectively overcomes pseudogene interference, demonstrating its suitability for large-scale carrier screening. However, its inherent limitation in detecting large gene rearrangements necessitates integration with comprehensive genetic counseling and confirmatory testing for specific genotypes in clinical practice.