The gray tiger longicorn beetle, Xylotrechus rusticus Linnaeus (Coleoptera: Cerambycidae) is a stem-boring pest that can inhibit not only the transportation of nutrients in the trunk but also the tree growth, increasing the risk of tree breakage and causing economic losses. It is distributed in China, Iran, Turkey, Russia, Korea, Japan, and Southern Europe. This study aimed to investigate selected Beauveria strains that could be used as entomopathogenic fungi for the biological control of this pest. The high-virulence strains were screened among the selected strains by cumulative mortality, correct mortality, and lethal time to 50% mortality (LT50). These screened high-virulence strains were Bb01, CFCC83486, and CFCC81428. Bb01 exhibited 96.96% cumulative mortality, with an estimated LT50 of 3.28 days. CFCC83486 and CFCC81428 caused 89.29% and 75.74% cumulative mortality, with an estimated LT50 of 3.45 and 4.28 days, respectively. Pathogenicity at different concentrations and lethal concentration of 50% (LC50) of these high-virulence strains were investigated. The pathogenicity was found to be positively correlated with suspension concentration, and LC50 was negatively correlated with infection time. These suspensions of high-virulence strains at different concentrations were also investigated in the forest by brushing the suspensions on the poplar tree trunk infested with X. rusticus L. larvae. The most effective strain was found to be Bb01, whose cumulative mortality reached 76.33% at 1.32 × 10 8 conidia mL −1, followed by the strain CFCC83486, whose cumulative mortality reached 65.17% at 1.32 × 10 8 conidia mL −1. This study provides an important basis for using B. bassiana in the biological control of X. rusticus L.