Hereby we present evaluation of high-resolution melting for mutation scanning applied to the cystic fibrosis transmembrane conductance regulator gene. High resolution melting was used for mutation scanning of selected samples derived from cystic fibrosis patients with a known cystic fibrosis transmembrane conductance regulator genotype. We tested 19 different disease-causing cystic fibrosis transmembrane conductance regulator mutant genotypes located within six exons of the cystic fibrosis transmembrane conductance regulator gene (4, 7, 10, 11, 14b and 22). Normalized melting curves of tested samples were compared to sequenced-verified wildtype samples. Determined mutations are as follows: p.F508del, p.I507del, p.G551D, p.R347P, c.17171G>A, c.621+1G>T, p.Y122X, p.I336K, p.R553X, c.2789+5G>A, c.574delA, c.1811+1G>C, p.L1335F, p.L1335P, p.L1324P and p.M470V and represent minimally 76.5 % of all cystic fibrosis alleles detected in the Czech cystic fibrosis population. All analysed samples with mutant genotypes were unambiguously distinguished from wild-type samples. High-resolution melting analysis enabled reliable detection of all single-nucleotide polymorphism classes and 1or 3-base pair deletions. We examined the specificity, sensitivity and precision of this methodology. High-resolution melting analysis is an economical, sensitive and specific close-tube method and has a high utility for the detection of unknown mutations in cystic fibrosis DNA diagnostics.