Objective?To explore the feasibility and application value of using high resolution melting curve technology to screen hot-spot mutations of pathogenic genes in 21 hydroxylase deficiency.?Method?Aimed at the four hot-spot mutations on?CYP21A2?gene, i.e., c.293-13A/C>G(I2G), c.518T>A(p.I173N), c.955C>T(p.Q319X) and c.1069C>T (p.R357W), the corresponding specific primers were designed. High resolution melting curve technology was used and DNA sequencing was combined to verify the results.?Results?High-resolution melting curve technique could definitely distinguish the four hot-spot mutations in?CYP21A2?gene and the results were consistent with Sanger sequencing showing fine correctness. The repeated analyses of three times showed that the coefficients of variance of Tm value were very small (0.168% to 0.620%).?Conclusion?The high-resolution melting curve technology as an efficient, accurate and low-cost screening method could be used for screening hotspot mutations on?CYP21A2?gene in healthy or high-risk population with certain clinical application value.