Abstract: In this study，based on the‘Ziyingui’DNA and 2 × Es TaqMasterMix，the SCoT-PCR amplification system of Osmanthus fragrans was established by optimizing reaction annealing temperature，the amount of template DNA，primer concentration and other factors. This system contained 30 ng template DNA，0.4 μmol ? L-1 primers in 12 μL mixture，and the most suitable annealing temperature of different primers was 48，49.9，54.3 and 56 ℃ respectively. Twelve Osmanthus fragrans cultivars were analyzed using this system，and the amplified bands were very clear. The 12 primers generated a total of 244 fragments from 12 Osmanthus fragrans cultivars，and 97.47% of fragments showed polymorphic. At the level of similarity coefficient 0.57，‘Jiulonggui’was to one group and other cultivars were to anothergroup. In the second group，‘Jingui’，‘Zaoyingui’and‘Ziyingui’were in one subgroup，and the other eight cultivars were in another subgroup. Our results indicated that genetic relationship between color and variety was not perfectly correlated，which is consistent with the results of other molecular markers.

Key words: Osmanthus fragrans, SCoT molecular marker, PCR system optimization, genetic analysis