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RAPD analysis was applied in studying the classification and genetic relationship of 12 cultivars of Gladiolus hybridus Hort. Twenty-six arbitrary primers screened from 80 primers were used for further PCR and diversity analysis. A total of 164 RAPD sites were detected with a mean of 6.31 fragments amplified for each primer. A total of 151 polymorphic DNA fragments were detected among all the 164 amplified fragments, which accounted for a high level of 92.8% of all and could be used for identification of different cultivars. The result revealed that the germplasm resource of Gladiolus hybridus Hort. cultivar had a narrow genetic base on molecular level.