Tramadol is a widely used, synthetic opioid analgesic for the prevention and treatment of moderate to severe pain in acute or chronic conditions. Several studies have been performed to evaluate its safety. Among the various possible toxic effects for this drug overdose is its potential genotoxicity. The purpose of the present study was to assess the possible genotoxic effects of tramadol dependence in humans using the chromosomal aberration (CA), sister chromatid exchange (SCE), and the micronucleus (MN) assays. This work was carried out on 30 adult male subjects ranged in age from 20 to 35 years; 10 of them were used as controls, 10 subjects had orally taken tramadol for 2-3 years, and another 10 subjects had orally taken tramadol for 4-5 years. Those poisoned subjects (No=20) were admitted to Tanta Poison Center in the period from June 2011 to January 2012 and were diagnosed as tramadol overdose on top of dependence. The results showed elevation in the frequencies of chromosomal aberrations, SCEs and micronuclei in cultured lymphocytes from tramadol-dependent patientswhich were related to the duration of drug intake.