Objective To study the influencing factors and quality control of chromosome preparation during chromosome aberration test in vitro, and to summarize and analyze the method and key points of successful preparation of chromosome specimens in vitro. Methods Chinese hamster lung cells (CHL) were used for cell culture and chromosome preparation. Mitomycin and cyclophosphamide were used as positive mutagens. After routine hypotonic treatment, fixation, and squash preparation, finally, to read the film under the microscope. Results The CHL chromosome aberration test showed that both the chromosome aberration rates of mitomycin- and cyclophosphamide-treated cells were significantly increased ( >20%), while the aberration rates in the negative control group were less than 5%, either with and without metabolic activation. The success rate was high and the prepared chromosomes were well dispersed with a moderate length. Conclusions Many factors can affect the specimen preparation in chromosome aberration test. Every step is very important, and it should be strictly following the operating procedures. It is of importance to grasp the principle of each step and to operate carefully, patiently and scientifically in order to prepare good specimens.