Hy droponics is a common soilless type of culture. It is clean and gives high-yield crop production. Lettuce is the most widely grown leafy vegetable in hydroponics. The Pythium species causes root rot, a serious disease in lettuce. In this study, we investigated an effective elicitor to promote resistance in lettuce after pathogen infection. To evaluate the plant defense gene response profile, the expression stability of five candidate genes was studied using RT-qPCR. The LsPR1 and LsLTC2 gene expression were higher in lettuce leaves treated with non-pathogenic Pythium PyASR23 than in roots compared to untreated plants. To assess the disease, the rate of relative gene expression of LsLTC2, LsPR-1b like, LsERF, LsAOS, and LsDEF was associated with Pythium root rot pathogenesis. As a result, it was revealed that alterations in these gene expressions were a defense response to Pythium infection, which increased after pre-treatment with various elicitors for 24 hours. When the chemically stimulated lettuce was infected with pathogenic PySR31, the expression of these genes increased dramatically. These results suggested that non-pathogenic PyASR23 might be employed as an elicitor in lettuce roots and contribute to disease control by activating plant defense gene expression against the Pythium pathogen.