Abstract: Objective To establish and compare two methods for chromosome preparation from LMH cells. Methods Twenty-four bottles of LMH cells were divided into two groups, one group was treated by 0.1% gelatin before cells subcultured, the other group was cultured directly in the bottle. After 72 hours, in-situ method was used for preparation of karyotypes of cells in gelatin group, and the other group was prepared with drop method. Results There was no significant difference in chromosome numbers and relative length between in-situ method and drop method. While, more karyotypes were found with in-situ method and it costed less steps and seemed more stable when compared with drop method. Conclusion In situ method is superior to drop method for chromosome preparation from LMH cells.