Cystic fibrosis (CF) is the most common severe autosomal recessive disorder caused by a wide spectrum of mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The frequencies, types and distributions of mutations vary widely between different populations and ethnic groups. The aim of this study was to perform a comprehensive analysis of the CFTR gene in an Iranian heterogeneous population. 20 CF patients diagnosed according to clinical evaluation and elevated sweat chloride value and presenting no common CFTR mutation, were analyzed for mutations and polymorphisms in exons 7, 20 and 21 and parts of introns 6, 7, 19, 20 and 21 of CFTR gene using PCR-sequencing. Sequencing of exon 7 revealed the presence of two variations c.864G>A (rs766189605) and c.910C>T (rs121909011) with the frequencies of 10% and 2.5%, respectively. c.864G>A is a synonymous variant that happens in amino acid valine 318 (GTG/GTA) and c.910C>T is a pathogenic missense variant that occurs at amino acid 334 (R334W) of CFTR protein leading to the change of arginine to tryptophan. Two variations c.3780A>G (rs1800130) and c.3783+117T>G (rs10155917) were also found in exon 20 and intron 20 with the frequencies of 7.5% and 5%, respectively. No mutation or polymorphism was found in exon 21. Ascertainment of CFTR mutation carrier frequencies and CF incidence among heterogeneous Iranian populations seems to be a necessity.