Carrier screening for monogenic diseases is becoming increasingly important in preventive medicine, yet selecting appropriate target genes remains a complex task, especially in countries with significant ethnic and geographic diversity such as China. This study aimed to develop a strategy to screen for carrier screening target genes suitable for the Chinese population, considering regional variations in carrier frequencies (CFs). We analyzed a dataset from a large-scale, multicenter carrier screening study, encompassing 33,104 individuals from different regions of China and carrier status for 223 genes. We focused on the CFs of these genes across regions. The study first stratified the population based on participants' self-reported ancestral places and then applied consensus k-means clustering analysis to the CF characteristics of these regions. This approach enabled us to identify distinct regional subpopulations with shared genetic backgrounds. The results showed that the regions clustered into three subpopulations (North, South, and Far South) based on CF characteristics, and 44 genes exhibited significant CF differences across these subpopulations (α=0.05). Applying an overall CF threshold without considering regional diversity would have excluded 11 regionally prevalent genes from the screening panel. By incorporating regional variations, we accurately identified 58 genes that met the recommended CF criteria (autosomal gene CF >
1/200, X-linked gene CF >
1/40,000) in at least one subpopulation. This study emphasizes the importance of considering regional diversity when designing carrier screening panels for monogenic diseases in China. Our proposed strategy, combining regional stratification and clustering analysis, provides a more precise method for selecting target genes, thereby enhancing the effectiveness and relevance of screening programs across different Chinese populations.