Background: Addiction is a complex disease influenced by genetic, environmental, developmental and social factors; altered serotonin transmission is thought to increase susceptibility to a wide range of substance use disorders. Serotonin transporter (5HTT) gene polymorphisms were found to be associated with some forms of drug abuse. Aim of the work: the aim of this study was to examine an association between 5-HTTVNTR genotype variants in a serotonin transporter gene and susceptibility to tramadol dependence and to examine the correlation between the aforementioned genotype variants and the comorbid psychiatric conditions in tramadol abuse patients. Subjects and Methods: A case–control gene study was conducted to evaluate the frequencies of 5-HTTVNTR genotype variants using SYBR green real-time PCR with melting curve analysis in tramadol addict and control subjects. Sixty tramadol addict subjects (30 with comorbid psychiatric condition and 30 without comorbid psychiatric condition) were taken from Outpatient Clinic of Kasr Al Ainy Hospital and inpatient addiction ward. The control group consisted of thirty apparently healthy subjects with no history of drug use or current use. Results: The less efficient transcriptional genotypes for 5-HTT polymorphisms were more frequent in tramadol addicts, while there were no statistically significant differences between both addict groups. Also, mutant "10" allele frequency was highly significantly increased in tramadol addicts than control group and in tramadol with comorbid psychiatry group than tramadol without comorbid psychiatry group. Conclusion: in this study an association was detected between the presence of 5-HTTVNTR with less transcriptional efficient genotypes and tramadol addiction and these genotypes increases the likelihood of the association of psychiatric comorbidity with tramadol dependence, which suggests that the alteration of the serotoninergic system might be implied in psychiatric problems in tramadol dependence.