Background: In the phase II KEYNOTE-199 study (NCT02787005), pembrolizumab monotherapy demonstrated antitumor activity in pts with docetaxel-refractory mCRPC (n = 258). Here we evaluated the association between prespecified molecular biomarkers and clinical outcomes. Methods: Cohorts 1 (C1) and 2 (C2) enrolled pts with RECIST-measurable PD-L1–positive (combined positive score [CPS] ≥1 using immunohistochemistry) and PD-L1–negative (CPS <1) disease, respectively. C3 enrolled pts with nonmeasurable, bone-predominant disease, irrespective of PD-L1 status. Biomarkers evaluated in this analysis were tumor mutational burden ([TMB; mutations/exome] n = 155), PD-L1 CPS (n = 255), tumor microenvironment–based 18-gene RNA expression profile ([GEP] n = 196), and microsatellite instability ([MSI] as determined by Promega PCR analysis; n = 147). Outcomes evaluated for C1 and C2 (n = 200) were ORR, disease control rate (DCR), and radiographic PFS (rPFS) per blinded, independent central review per PCWG-modified RECIST v1.1. Outcomes evaluated for C1-C3 (n = 258) were prostate-specific antigen (PSA) response, time to PSA progression, and OS. Significance of continuous biomarkers (CPS; TMB; GEP) was prespecified at 0.05 for one-sided P values from logistic (ORR; DCR; PSA response) and Cox proportional hazard regression (rPFS; OS; PSA progression) adjusted for Eastern Cooperative Oncology Group performance status. Binary biomarkers (MSI) were analyzed using Fisher’s exact test (ORR; DCR; PSA response). Clinical data cutoff date: Jun 24, 2019. Results: Median TMB was 53.0 (interquartile range [IQR], 40.5 to 78.0), median CPS was 1 (IQR, 0 to 5), and median GEP was –0.64 (IQR, –0.88 to –0.46); 6 pts (2.3%) had MSI-high tumors. In C1-C3, TMB was associated with PSA response (one-sided nominal P = 0.0016) and time to PSA progression (one-sided nominal P = 0.00092). In C1-C3, PD-L1 CPS was associated with PSA response (one-sided nominal P = 0.046) and time to PSA progression (one-sided nominal P = 0.021). In C1-C3, GEP was not significantly associated with response. In C1-C3, MSI was associated with PSA response (one-sided nominal P = 0.019). Conclusions: In this biomarker analysis from KEYNOTE-199 C1-C3, TMB and PD-L1 CPS were associated with better PSA response; however, small pt numbers limit definitive conclusions on ORR, DCR, and OS. Further evaluation of molecular biomarkers in pts with mCRPC treated with pembrolizumab is warranted. Clinical trial information: NCT02787005.