Abstract: The study aims to establish an optimal reaction system of SCoT- PCR in blueberry and provide technical support for molecular breeding and genetic diversity analysis. Blueberry was used as material, orthogonal design method was applied to optimize SCoT-PCR amplification system of blueberry in five factors such as Mg²⁺, dNTPs, Taq DNA polymerases, template DNA and primers at five levels. An optimal SCoT-PCR amplification system (20μL) of blueberry was established, i.e. Mg²⁺ 2.813 mmol/L, dNTPs 0.100 mmol/L, Taq polymerase 1.5 U, primer 0.2μmol/L, template DNA 10 ng. 32 SCoT primers and six blueberry varieties‘( Centurion’,‘Premier’,‘Homebell’, O'Neal’,‘Tifblue’,‘Misty’) were analyzed with the optimized reaction system. The study indicated that amplified results with fine diversity and clear strap were gained.