Abstract: The main factors affected ISSR reaction were tested with Miduzipi garlic as test materials to optimize ISSR amplification system .In this research, the orthogonal design was used to optimize ISSR-PCR system on pepper in for 4 levels of 5 factors (Taq DNA polymerase, primer concentration Mg2+, dNTPs and DNA template) respectively. The results showed that: the orthogonal experiment design was a practicable method and in a total volume of 25 μL ISSR-PCR system, the optimum reaction system of ISSR-PCR was 10×PCR Buffer 2.5 μL, 2.5 mmol Mg2+, 1.5 U Taq DNA polymerase, 0.15 mmol dNTPs, 0.4 μmol primer and 20 ng template DNA. The ISSR amplifications for 20 cultivars of common garlic were then investigated through the above reaction system. The system was completely suitable to analyze the genetic information for different garlic cultivars and thus supplied the further genetic diversity of garlic with theoretic bases.