Abstract: To screen the optimum ISSR-PCR reaction system for potato, the experiment was conducted by using orthogonal design with 4 levels of 5 factors (i.e. rTaq DNA polymerase, Mg2+, DNA, dNTPs and primers). Results showed that the optimal reaction system of ISSR-PCR was as follows: 0.5 U rTaq DNA polymerase, 2.5 mmol/L Mg2+, 75 ng DNA, 200 mol/L dNTPs and 0.8 mol/L primer in 25 L reaction volume. And the PCR reaction effects was in the order of Mg2+ > rTaq DNA polymerase> dNTPs = primer> DNA. Then, 39 ISSR primers were screened by using the optimal reaction system, and 15 primers with clear band, more band number and good repeatability were selected. Finally, the optimized annealing temperatures of the 15 primers were obtained by gradient PCR.