Background: The effects of non-amplification short variant (SV) mutations in CD274 (PD-L1) on PD-L1 protein expression and immune checkpoint inhibitor (ICPI) therapy are unknown. Here, we present a retrospective analysis of CD274 mutations detected by comprehensive genomic profiling (CGP) and correlate these results with tumor-cell PD-L1 immunohistochemistry (IHC)-based expression assessment to better understand the relationship between mutations and protein expression of PD-L1. Methods: FoundationOne CGP was performed on hybridization-captured, adaptor ligation-based libraries using DNA and/or RNA extracted from 314,631 tumor samples that were sequenced for up to 406 cancer related genes and select gene rearrangements. PD-L1 IHC was performed on a subset of cases (n = 213) using the DAKO 22C3 PD-L1 IHC assay and scored with the tumor proportion score (TPS). Results: Overall, the prevalence of CD274 SV mutations was low (0.3%, 1,081/314,631) with 577 unique variants. The most common CD274 SV mutations were R260H (n = 51), R260C (n = 18), R125Q (n = 12), C272fs*13 (n = 11), R86W (n = 10), and R113H (n = 10). The prevalence of CD274 mutations varied depending on tumor type with diffuse large B-cell lymphoma (1.9%, 19/997), cutaneous squamous cell carcinoma (1.6%, 14/868), endometrial adenocarcinoma (1.0%, 36/3740), unknown primary melanoma (0.9%, 33/3679), and cutaneous melanoma (0.8%, 32/3874) having the highest frequency of mutations. Ultraviolet exposure was likely a mechanism for CD274 SV mutations in cutaneous tumors with high frequencies of ultraviolet mutational signatures (cutaneous squamous cell carcinoma [84.6%, 11/13], cutaneous melanoma [93.8%, 30/32], and unknown primary melanoma [100%, 32/32]), and microsatellite instability (MSI) was likely a mechanism for development of CD274 mutations in non-serous endometrial adenocarcinoma. Of the R260H cases concurrently tested with PD-L1 IHC, most (81.8%, 9/11) had no PD-L1 expression, which contrasts to the five E237K cases where most (80%, 4/5) had PD-L1 expression. This difference in protein expression of these two mutations was significantly different (p = 0.036). It was notable that nearly all samples (88.9%, 16/18) with a clonal truncating variant (nonsense or frame shift indel) and PD-L1 testing showed a PD-L1 TPS score ≤1, whereas three of four samples with sub-clonal truncating variants had TPS scores ≥5. Conclusions: We defined the landscape of CD274 mutations in a large cohort of tumor types that can be used as a reference for examining CD274 mutations as potential resistance biomarkers for ICPI. Furthermore, we presented novel data on the correlation of CD274 mutations and PD-L1 protein expression, providing important new information on the potential functionality of these mutations and can serve as a basis for future research.