We investigated whether polymorphisms of the endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), and GTP cyclohydrolase I (GTPCH) genes are involved in the aggravation of migraine induced by overuse of medications. We studied 47 patients with migraine (six males and 41 females; 36.4 ± 10.3 years of age) and 22 patients with migraine exhibiting medication overuse headache (MOH, one male and 21 females; 39.6 ± 9.9 years of age). The genotypes of polymorphisms of the eNOS (rs1799983), nNOS (rs2682826), and GTPCH (rs841) genes were analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The genotypic distributions of rs2682826 (T/T plus T/C vs. C/C, P = 0.254), rs1799983 (G/G vs. G/T plus T/T, P = 1.000), and rs841 (T/T plus T/C vs. C/C, P = 0.149) were not significantly different between patients with migraine and patients with MOH. The results of this study showed an absence of association between the polymorphisms of eNOS, nNOS, and GTPCH genes and the complication of MOH in patients with migraine.