In the phase III KEYNOTE-061 trial (NCT02370498), pembrolizumab did not significantly improve overall survival versus paclitaxel as second-line therapy for gastric/gastroesophageal junction (GEJ) adenocarcinoma with programmed death-ligand 1 (PD-L1) combined positive score (CPS) ≥1 tumors. The association of tissue tumor mutational burden (tTMB) status and clinical outcomes was determined, including the relationship with CPS and microsatellite instability-high (MSI-H) status. In patients with whole exome sequencing (WES) data 420/592 (71%); pembrolizumab, 218; paclitaxel, 202, the association of tTMB with objective response rate (ORR; logistic regression), progression-free survival (PFS; Cox proportional hazards regression), and overall survival (OS; Cox proportional hazards regression) were measured using one-sided (pembrolizumab) and two-sided paclitaxel P values. tTMB was also evaluated using FoundationOne®CDx 205/592 (35%). Prespecified equivalent cut-offs of 175 mut/exome for WES and 10 mut/Mb for FoundationOne®CDx were used. WES-tTMB was significantly associated with ORR, PFS, and OS in pembrolizumab-treated (all P < 0.001) but not paclitaxel-treated patients (all P > 0.6) in univariate analysis. The area under the receiver operating characteristics curve for WES-tTMB and response was 0.68 95% confidence interval (CI) 0.56-0.81 for pembrolizumab and 0.51 (95% CI 0.39-0.63) for paclitaxel in univariate analysis. There was low correlation between WES-tTMB and CPS in both treatment groups (r ≤ 0.16). WES-tTMB remained significantly associated with all clinical endpoints with pembrolizumab after adjusting for CPS and with PFS and OS after excluding known MSI-H tumors (n = 26). FoundationOne®CDx-tTMB demonstrated a positive association with ORR, PFS, and OS in pembrolizumab-treated patients (all P ≤ 0.003) but not PFS or OS in paclitaxel-treated patients (P > 0.1). This exploratory analysis from KEYNOTE-061 is the first to demonstrate a strong association between tTMB and efficacy with pembrolizumab but not paclitaxel in patients with gastric/GEJ adenocarcinoma in a randomized setting. Data further suggest tTMB is a significant and independent predictor beyond PD-L1 status. •Second-line pembrolizumab showed a strong association between tTMB and efficacy in gastric and gastroesophageal cancer.•There was low correlation between WES-tTMB and PD-L1 CPS.•WES-tTMB was associated significantly with pembrolizumab PFS and OS after excluding known MSI-H tumors.•Findings with FoundationOne®CDx-tTMB were similar to those with WES-tTMB.