This is the first report of the HSD3B2 variants c.121G > T (p.E41*) and c.757T > G (p.Y253D), which expanded the variant spectrum of HSD3B2. LRS is a robust diagnostic tool for CAH, offering comprehensive detection of genetic variants, including large deletions and SNVs in both cis and trans forms. The gene discussed is HSD3B2; the disease is congenital adrenal hyperplasia.