The comet assay is a sensitive test for detecting DNA damages used as an instrument to compare the intensity of the genotoxic effects of colchicine. The Pattern and the index value of three parameters of comet assay (tail length, tail intensity, tail moment) were compared among the autotetraploids produced from three populations of diploid cross pollinated Medicago sativa spp. Caerulea and autotetraploid populations from five diploid self pollinated species by treatment with three concentrations of colchicine (0.1, 0.5, and 1%) and five alfalfa cultivars. Results have shown the same level of increases and pattern in the parameters of comet "among the induced tetraploids from the two categories of medics" in the concentration of 0.1 and 0.5% of colchicine. Also increasing colchicine concentration from 0.1 to 0.5% resulted in a more pronounced augmentation of comet parameters. Induced tetraploids resulted from the two mentioned colchicine concentrations showed increases in the value of the three comet parameters compared to the cultivated alfalfa cultivars and two diploid categories medic species so the pattern of parameters were not similar. At the concentration of 1% of colchicine, only two annual species produced tetraploids showing very pronounced augmentation of comet parameters in comparison with 0.1 and 0.5% colchicine concentrations. Changes in patterns and values of the three parameters in induced tetraploids compared to cultivated alfalfa and diploid medic species demonstrate differential effects of damages of colchicine from one concentration to another, then a new variability in each concentration change will be created.