Abstract: ObjectiveTechnique of chromosome preparation for polyploid watermelon was improved,conventional karyotype and fluorescence karyotype for polyploid watermelons were compared in order to provide reference for research on genetics of watermelon and establishment of physical genetic map of watermelon . MethodPolyploid watermelons were used as materials in the study. Based on traditional chromosome preparation technique , namely wall degradation-hypotonic flaming-drying method, pre-hypotonic and after-hypotonic were deleted, and re-fixation and flame smear were combined into one procedure. Giemsa stain and DAPI fluorescence stain were used to stain, and carried out conventional karyotype analysis and fluorescence karyotype analysis respectively. ResultThe optimized enzymolysis flaming-drying method was used to prepare chromosome of polyploid watermelons. Compared with the traditional technique , the optimized one simplified three procedures which were pre-hypotonic and after-hypotonic of KCL solution and re-fixation by Carnoy fixative. Thus the time was 1.0-2.0 h shorter. Production efficiency was improved, and samples with complete amount, good dispersion and clear configuration were obtained. Slides stained with DAPI could shorten the detection time for 0.5-1.5 h each slide, and were more easily to be detected metaphases with more clear centromere,long arm and short arm of chromosome than stained with Giemsa . ConclusionThe modified method can improve the efficiency of chromosome preparation of polyploid watermelon. Fluorescent karyotype analysis is more accurate and efficient than conventional karyotype analysis.