Serotonin is involved in several central nervous system functions including pain threshold, mood regulation and drug reward. Overuse of acute medications is commonly identified as a causative factor for medication overuse headache (MOH). Apparently, MOH shares with other kinds of drug addiction some common neurobiological pathways. The objective of this study is to assess the role of serotonin metabolism genes in the genetic liability to MOH. We performed a genetic association study using polymorphisms of five serotonin metabolism-related genes: serotonin transporter (5HTT), serotonin receptor 1A(5-HT1A), serotonin receptor 1B (5-HT1B), serotonin receptor 2A (5-HT2A) and serotonin receptor 6 (5HT6)genes. We compared 138 patients with MOH with a control sample of 117 individuals without headache and without drug overuse, and with 101 patients with migraine without aura but without drug overuse (MO). The genotypic and allelic distributions of all polymorphisms investigated didnot differ among the three groups. In conclusion, our studydoes not provide evidence that the 5HTT, 5-HT1A, 5HT1B,5HT2A and 5HT6 gene polymorphisms play a role in the genetic predisposition to MOH.