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Simple Summary: PD-L1 expression determines patients' eligibility for immunotherapy. Current sampling does not consider the heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC) within the primary tumor. Moreover, potential differences are not considered when comparing primary tumors and their associated metastases or local recurrences, hereby excluding potential responders to immunotherapy. Here, we investigated the inter-sample heterogeneity of PD-L1 expression by analyzing multiple samples from individual patients. Multisection staining revealed clinically relevant CPS changes, which would have potentially affected treatment decisions in 28.7% (intra-tumoral), 44.4% (tumor vs. metastases), and 61.5% (initial tumor vs. local recurrence) of patients, respectively, compared with single-region staining. Increased CPS in primary tumors and lymph node metastases were associated with improved 5-year overall survival. Our results suggest that multiple tumor sections should be evaluated in HNSCC patients when assessing PD-L1 expression prior to potential immunotherapy, particularly if the initial result was negative. For practical reasons, in many studies PD-L1 expression is measured by combined positive score (CPS) from a single tumor sample. This does not reflect the heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC). We investigated the extent and relevance of PD-L1 expression heterogeneity in HNSCC analyzing primary tumors and recurrences (LRs), as well as metastases. Tumor tissue from 200 HNSCC patients was immunohistochemically stained for PD-L1 and analyzed using image-analysis software QuPath v3.4 with multiple specimens per patient. CPS was ≥20 in 25.6% of primary tumors. Intra-tumoral heterogeneity led to a therapeutically relevant underestimation of PD-L1 expression in 28.7% of patients, when only one specimen per patient was analyzed. Inter-tumoral differences in PD-L1 expression between primary tumors and lymph node metastasis (LNM) or LR occurred in 44.4% and 61.5% (CPS) and in 40.6% and 50% of cases (TPS). Overall survival was increased in patients with CPS ≥ 1 vs. CPS < 1 in primary tumors and LNM (hazard ratio: 0.46 and 0.35; p < 0.005); CPS in LR was not prognostic. Our analysis shows clinically relevant intra- and inter-sample heterogeneity of PD-L1 expression in HNSCC. To account for heterogeneity and improve patient selection for immunotherapy, multiple sample analyses should be performed, particularly in patients with CPS/TPS < 1. [ABSTRACT FROM AUTHOR]