Abstract Background There is a growing relationship between PD-1/PD-Ls and autoimmune disease We attempted to explore the relationship of the PD-L2 expression in patients with thymoma, especially with Myasthenia Gravis (thymomatous MG). Methods Seventy patients with thymoma who underwent surgical resection between January 2017 to December 2018 were retrospectively reviewed. The PD-L2 expression was evaluated by immunohistochemistry. The association between the PD-L2 expression and the clinicopathologic features was investigated, especially thymomatous MG. Results The PD-L2 expression was positive in 41 patients (58.6%) and negative in 29 patients (41.4%). The PD-L2 expression was significantly associated with WHO histology of type B2 and B3 thymoma (p=0.008) and the status of MG (p=0.002). In addition, PD-L2 positive tumors showed a significantly smaller tumor size (P=0.017). Ectopic thymus was significantly more often seen in the PD-L2 positive group (p=0.035). The patients with MG (p=0.001) and WHO type B2 and B3 (p=0.007) have significantly higher PD-L2 scores. Multivariate logistic regression model showed the status of MG (OR 12.601, 95%CI 2.406-65.995, p=0.02) and age (OR 0.199, 95%CI 0.046-0.863, p=0.031) were significantly associated with the expression of PD-L2. The analysis of 33 patients with MG shows age was not associated with the expression of PD-L2 (OR 0.04, 95%CI 0.001-3.2, p=0.15). Conclusions A strong expression of PD-L2 in thymoma was significantly associated with thymomatous MG and WHO histologic type B2 and B3. In addition, PD-L2 may play a potential role in the pathogenesis of thymomatous MG.