Description: Purpose: Understanding why some melanomas test negative for PD-L1 by IHC may have implications for the application of anti-PD-1 therapies in melanoma management. This study sought to determine somatic mutation and gene expression patterns associated with tumor cell PD-L1 expression, or lack thereof, in stage III metastatic melanoma to better define therapeutically relevant patient subgroups. Experimental Design: IHC for PD-L1 was assessed in 52 American Joint Committee on Cancer stage III melanoma lymph node specimens and compared with specimen-matched comprehensive clinicopathologic, genomic, and transcriptomic data. Results: PD-L1–negative status was associated with lower nonsynonymous mutation (NSM) burden ( P = 0.017) and worse melanoma-specific survival [HR = 0.28 (0.12–0.66), P = 0.002] in stage III melanoma. Gene set enrichment analysis identified an immune-related gene expression signature in PD-L1–positive tumors. There was a marked increase in cytotoxic T-cell and macrophage-specific genes in PD-L1–positive melanomas. CD8A high gene expression was associated with better melanoma-specific survival [HR = 0.2 (0.05–0.87), P = 0.017] and restricted to PD-L1–positive stage III specimens. NF1 mutations were restricted to PD-L1–positive tumors ( P = 0.041). Conclusions: Tumor negative PD-L1 status in stage III melanoma lymph node metastasis is a marker of worse patient survival and is associated with a poor immune response gene signature. Lower NSM levels were associated with PD-L1–negative status suggesting differences in somatic mutation profiles are a determinant of PD-L1–associated antitumor immunity in stage III melanoma. Clin Cancer Res; 22(15); 3915–23. ©2016 AACR .