Hibiscus rosa-sinensis L is an evergreen plant commonly found in the tropical and subtropical areas. H.rosa-sinensis has the great diversity in the petal shape, petal colors, plant habits, and even the chromosome numbers. The information of chromosome number is important in cytology and can further be explored for the study of its morphology and physiology. The H.rosa-sinensis chromosome number has been reported to vary from 46, 54, 63, 68, 72, 77, 84, 90, 96, 112, 132, to 144, making the determination of the basic number very difficult. The overlapping chromosome samples generally gave the bias data for chromosome counting. The optimum sample preparation will result in the good chromosome appearance which will provide the clear image to be counted and avoid the bias. One of the steps in the preparation method for chromosome observation is the pretreatment of the sample. For this step, paradichlorobenzene, 8-hydroxyquinoline, and cool water can be used. However, there is no clear report optimizing the pretreatment process. Thus in this study, we optimized the preparation method for chromosome observation by comparing those different pretreatment methods. Different chemical (paradichlorobenzene, 8-hydroxyquinoline, and cool water) and different pretreatment lengths from 0 to 24 hours were compared. After each treatment, samples were observed and the number of cells showing each phase of the cell cycle (interphase, early prophase, late prophase, metaphase, anaphase, telophase) was counted. Specific chemical and pretreatment time display the most cells in late prophase and metaphase chromosomes were selected to be the most optimum condition. According to the data obtained, 3 hours treatment of the samples using cool water resulted in the most optimum condition for chromosome observation. The optimum preparation method will give further insight into the chromosome study of the H. rosa-sinensis.