Tomato of known genotype was used in the study to identify tomato Ty-3 gene, I-2 gene and Mi gene,which tightly linked to tomato yellow leaf curl virus disease-resistant gene Ty-3, tomato Fusarium wilt resistantgene I-2 and tomato root-knot nematode resistant gene Mi, respectively. We established a multi-PCR methodwhich can be utilized to identify these resistant genes in tomato simultaneously with the three SCAR makers. Afterseveral times tests, the PCR products were completely correspond to the amplified bands produced by single SCARprimer. The consistent results showed that three resistant genes could be identified simultaneously by using corre-sponding primers under the adapted amplification conditions. We screened 300 different tomato germplasm usingthe multi-PCR method. The results showed molecular detection was the same as vaccination identification. The tech-nical procedure is very simple, efficient and fast. It could be useful for molecular marker assisted breeding andscreening of tomato resistant germplasm.