PD-L1 is expressed in a percentage of lung cancer patients and those patients show increased likelihood of response to PD-1 axis therapies. However, the methods and assays for the assessment of PD-L1 using immunohistochemistry are variable and PD-L1 expression appears to be highly heterogeneous. Here, we examine assay heterogeneity parameters toward the goal of determining variability of sampling and the variability due to pathologist-based reading of the immunohistochemistry slide. SP142, a rabbit monoclonal antibody, was used to detect PD-L1 by both chromogenic immunohistochemistry and quantitative immunofluorescence using a laboratory-derived test. Five pathologists scored the percentage of PD-L1 positivity in tumor- and stromal-immune cells of 35 resected non-small cell lung cancer cases, each represented on three separate blocks. An intraclass correlation coefficient of 94% agreement was seen among the pathologists for the assessment of PD-L1 in tumor cells, but only 27% agreement was seen in stromal/immune cell PD-L1 expression. The block-to-block reproducibility of each pathologist’s score was 94% for tumor cells and 75% among stromal/immune cells. Lin’s concordance correlation coefficient between pathologists’ readings and the mean immunofluorescence score among blocks was 94% in tumor and 68% in stroma. Pathologists were highly concordant for PD-L1 tumor scoring, but not for stromal/immune cell scoring. Pathologist scores and immunofluorescence scores were concordant for tumor tissue, but not for stromal/immune cells. PD-L1 expression was similar among all the three blocks from each tumor, indicating that staining of one block is enough to represent the entire tumor and that the spatial distribution of heterogeneity of expression of PD-L1 is within the area represented in a single block. Future studies are needed to determine the minimum representative tumor area for PD-L1 assessment for response to therapy.