Using five cassava cultivars as explants,different colchicine concentrations (0.1,0.3,0.5 g/L) and time duration of treatment (3,6,9 h) were combined to induce autotetraploids of cassava in vitro using axillary buds.Generated shoots were transferred onto the basic MS medium for further development.Ploidy analysis via both flow cytometry and root tip squash technique verified that recovered plantlets from all five cultivars were stable autotetraploids after several subcultures.Autotetraploid plantlets originated from Col22 and SC8 and its corresponding parental plants were transferred into greenhouse.Phenotypes of autotetraploids were comparatively analyzed after five-month growth.Results showed that leaves of autotetraploids became significantly wider and thicker than those of parental plants.The length and width and density of stomas had significant difference between the control and autotetraploids.These novel autotetraploids have great potential for the further genetic improvement of cassava.