Prostaglandin I2 receptor plays a major physiologic role in the relaxation of arterial smooth muscle and vasodilation and possibly during migraine attacks. Therefore, in this study, the coding and noncoding exons and exon-intron boundaries of Prostaglandin I2 receptor gene were examined in patients with migraine headache and healthy controls and the potential effects of identified single nucleotide variations were evaluated using direct PCR-sequencing and in silico analysis. In this study, the peripheral blood samples of 50 patients and 50 controls were examined to find any mutation in coding and noncoding exons and exon-intron boundaries of PTGIR gene. DNA was extracted and all the samples were amplified by polymerase chain reaction (PCR) and sequenced. In this study, the patients had a mean age of 35.235 ± 10.99 years (range, 9-60 yrs.), and female to male ratio was 4:1 in this group. The controls had a mean age of 35.058 ± 11.116 years (range, 8-59 yrs.), and female to male ratio was 3.7:1.3 in this group. Two patients had mutations in exon 2. The first mutation was located in exon 2 (at amino acid position 251) of PTGIR gene at nucleotide position c.866A > T, a synonymous variant described previously in the database. The second mutation was located in exon 2 c.867G > A, which is a missense variant. Sequence analysis revealed high occurrence of previously reported intronic variants mostly in a homozygous statue. The data supported the hypothesis that mutations in PTGIR gene, particularly the mutation we described, should be considered even in cases of migraine. The presence of this mutation in patients with family history raises important issues regarding genetic counselling.