Abstract: By using the specific primers designed on the basis of conserved sequences of coat protein (CP) gene, a multiplex PCR protocol for the detection of three main viruses (i.e., Freesia mosaic virus, FreMV, Cucumber mosaic virus, CMV and Bean yellow mosaic virus,BYMV) in freesia was developed. Three specific fragments were simultaneously amplified in a single PCR reaction. Their molecular weights were determined to be 340 bp, 628 bp and 212 bp. The sequence analysis indicated that the three viruses shared at least 97 percent homology with other related viruses. The method’s sensitivity was shown to be capable of positively identifying the three viruses with≥10-2 mg of freesia tissue sample.