Abstract: To obtain desirable chromosome samples for studying wheat roots, two preparation methods were compared for optimization. The root-tip cells of three wheat varieties (i.e., Taichung 29, Chinese Spring and Zhong 4) were collected for the study on the conventional and the enzymatic digestion methods. Aiming for microscopically clearly defined chromosome samples, the evaluation and optimization criteria encompassed the operation time, staining procedure, chromosome dissociation and sample preparation of the methods. The conventional method took up to a week to operate, and required precise controls in dissociating and staining the chromosomes as well as preparing microscope slides. On the other hand, the enzymatic method was conducted by pulverization without staining and slice-making, and the entire procedure could be completed in 4-5 days. Not only that the procedures were simpler and less prone to errors, the enzymatic method could generate many specimens in a single run. Consequently, it was considered superior to the conventional method for the chromosome sample preparation.