Abstract: In this research,triplex PCR systems were established and optimized to specifically detect Cucumber mosaic virus(CMV) and Lily symptomless virus(LSV) of two main Lanzhou lily viruses with 18S ribosomal RNA used as an internal control.Results showed that an Annealing temperature of 52.5°C,Taq DNA polymerase of 0.025 U/μL,dNTP of 0.6 mmol/L,Mg²⁺ of 4 mmol/L,three primer pairs of 0.4 μmol/L,and < 30 cycles were the optimal amplification conditions for triplex PCR systems.Differences in viruses from different parts of Lanzhou lily were detected by the optimal triplex PCR systems.The intensity of specific amplified bands of LSV from the different sample parts was more uniform than the intensity of CMV,and the relative contents of CMV was maximum outside of the bulb for the whole viruses infected plants.Our results provide technical support for the detection of virus and virus-free tissue culture and rapid propagation of Lanzhou lily.