In this study, combined with Rosaceae database, through multiple sequence alignment, expression analysis and validation, we screened the defenserelated Marker genes and corresponding primers in the genome of apple (Malus domestica) and pear (Pyrus bretschneideri). We took the suspension cells of Duli and DongbeiShanjingzi as materials, treated with 20% metabolites of Valsa canker (Vp), and analyzed the expression patterns of marker genes in response to Vp signal by realtime fluorescent quantitative PCR (RT qPCR), which lays a foundation for the rapid detection of disease resistance reaction of apple and pear and systematic study of resistance mechanism to Vp of Duli.The results showed that: (1) a total of 15 Marker genes and corresponding primers for salicylic acid (SA), jasmonic acid (JA), ethylene (ET), systemic acquired resistance (SAR) response, pathogenassociated molecular patternprimed immune response (PTI), phycocyanin and defenserelated signals were screened by search and comparison. (2) The Cq values ranged from 18 to 25 at the cDNA concentration of 100 ng·μL^-1 for each treatment, and the PCR amplification efficiency was high, which indicated that the primers of the 15 Marker genes could accurately reflect the activation of the resistance response and could be used as Marker genes for the respective signaling pathways. (3) Compared with control, the accumulation of ROS in suspension cells of Duli after Vp metabolite treatment showed a significant increase with the treatment time, and the value of ROS signal reached 3.2 times of the control at 6 h, show that Vp metabolite activates the plant immune response (PTI), which induce the burst of ROS. (4) RT qPCR analysis showed that Marker genes of SA, JA, PTI, SAR, Rgene, phytogenesis and defenserelated signaling pathways were upregulated in Duli suspension cells after Vp metabolite treatment, among which, expression of SA, JA and SAR related genes ChiV (Chitinase class V), LOX1 (Lipoxygenase 1), PR4 (PathogenesisRelated 4), and defense response related gene PAL1 (Phe Ammonia Lyase 1) were robustly induced, the highest levels climbed to 1718-, 691-, 1072- and 6369fold of the control, respectively. The results shown that it mainly activated genes of SA, JA, SAR and defenserelated signaling pathways when Duli subjected to Vp metabolite, and then resist the further invasion of pathogens which in turn were able to resist further invasion of the pathogen. It was found that SA, JA, SAR and defense response signals were involved in the response of Duli to Vp, thereby improving its disease resistance.