Background: Methotrexate is an antineoplastic, antipsoriatic and antirheumatic agent belongs to the group of antimetabolites and inhibits folic acid metabolism. Materials and methods: To investigate its possible effect, sixty male mice were randomly assigned to one of four groups (one control and three treated groups with different doses of methotrexate). Mice of groups 1, 2 and 3 were intraperitoneally injected with 2.5, 5 and 10 mg /kg b.wt. methotrexate respectively. All the control and treated animals were sacrificed at 24, 48 and 72 hour by cervical dislocation post treatment. Results:Micronucleus assay results showed that methotrexate treatment induced genotoxicity in bone marrow cells, the number of micronucleated polychromatic erythrocytes (MNPCEs) and the ratio of polychromatic erythrocytes / normochromatic erythrocytes was gradually increased significantly  (P < 0.001) by increasing dose and time of treatment in methotrexate treated groups in comparison with the control group. An analysis of randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR) showed different ranges of DNA modifications in the treated groups after 24, 48 and 72 hour of treatment in comparison with the control group. Results of this study indicate that methotrexate treatment induced cytotoxic and genotoxic effect on bone marrow cells and DNA content of male albino mice even after a low dose and single treatment. Conclusion:  Therefore, the therapeutic uses of methotrexate should be restricted to a very narrow range border.