Background: Previous studies have indicated genetic alterations (GA) of DDR genes and tumor mutational burden (TMB) as potential markers for anti-PD-1/PD-L1 therapy in several cancer types. The study explored whether GA of DDR genes and TMB are associated with clinical benefit (CB) for ESCC patients receiving anti-PD-1/PD-L1 therapy. Methods: Thirty-five ESCC patients treated with PD-1/PD-L1 blockade antibody, alone or in combination, were enrolled. Tumor response was evaluated per RECIST 1.1, and CB was defined as complete response, partial response or stable disease at least 6 months. Formalin-fixed paraffin-embedded ESCC tissues were analyzed by FoundationOne CDx. All loss-of-function alterations were considered as deleterious according to previous reports (Teo MY, et al: Clin Cancer Res 2017; 23:3610-8 and J Clin Oncol 2018; 36:1685-94). Results: All of 35 enrolled patients was male: 16 and 19 received PD-1/PD-L1 blockade alone and PD-1/PD-L1-based combination therapy, respectively. The response rate was 14%, and the CB rate was 29%. All patients had GA of TP53 (100%), followed by CDKN2A/B (71%), and FGF3, FGF4 plus FGF19 (40%). The median TMB was 4 muts/Mb (range 1-16) and all evaluable ESCC tissues (N=31) were microsatellite stable. Twenty-one (60%) patients had GA of DDR genes, but only 4 (11%) patients had deleterious GA of DDR genes. The clinicopathological characteristics were not significantly different between patients with or without GA of DDR genes or those with or without deleterious GA of DDR genes. Patients harboring deleterious GA of DDR genes trended to have improved response rate (25% vs. 12.9%, P = 0.063), CB rate (50% vs. 22.6%, P = 0.268), and median PFS (4.1 vs. 1.8 mon, P = 0.074). Neither TMB nor all GA of DDR genes was associated with response or CB. Conclusions: Deleterious GA of DDR genes may be associated with therapeutic efficacies of anti-PD-1/PD-L1 therapy in patients with ESCC. (Funded by NHRI-107A1-CACO-01181816, NHRI-108A1-CACO-01191916, MOST-106-2314-B-002-225-MY2, NTU-108L901403, and MOST 108-3017-F-002-004-).