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Background Tumor Mutational Burden (TMB) has emerged as a predictive biomarker for immune checkpoint inhibitor (ICI) response across multiple cancer types. However, current clinical TMB assessments often rely on targeted panels with limited genomic coverage, leading to variability in TMB estimation and potential misclassification of patients. Target-enhanced whole genome sequencing (TE-WGS) provides an expanded, genome-wide approach that may improve the accuracy and consistency of TMB measurements, particularly for immunotherapy decision-making.Methods We analyzed 202 tumor samples from lung, breast, colorectal, ovarian, and fallopian tube cancer patients. TMB was evaluated using the validated large targeted panel tests (TP), such as TruSight Oncology (TSO500) and GeneseeqPrime Pan-Solid Tumor, and CancerVision’s target-enhanced WGS (TE-WGS). TP define TMB as non-synonymous somatic mutations per megabase (mut/Mb) of the panel’s genomic regions, with high TMB classified as ≥10 mut/Mb. In contrast, TE-WGS calculates TMB across the effective genome size (2.9Gb) without limiting to coding regions. TE-WGS was analytically validated, demonstrating 99.8% sensitivity for single nucleotide variants (SNVs), 99.2% for indels, and high positive predictive values (SNVs: 99.3%, indels: 98.7%).Results TMB values were compared between TE-WGS and TP. The TE-WGS assay showed strong concordance with TP for high (≥10 mut/Mb) and low ( < 10 mut/Mb) TMB classification: Sensitivity 79.3% (95% CI: 61.6–90.2%) and Specificity 95.4% (95% CI: 91.1–97.6%). Of the 202 samples, 23 (11.4%) were concordantly classified as high TMB, and 165 (81.7%) as low TMB. Discordance was observed in 14 cases (6.9%): 8 high TMB by TE-WGS but low by TP, and 6 low TMB by TE-WGS but high by TP. Analytical evaluation revealed that TE-WGS quantification was robust across varying tumor cellularity levels, demonstrating consistent performance independent of tumor purity. Discordant cases highlighted the advantage of TE-WGS in capturing mutations outside the genomic regions covered by TP, underscoring the importance of genome-wide assessment for accurate TMB quantification.Conclusions TE-WGS offers a more comprehensive and reliable approach to TMB assessment compared to targeted panels, minimizing limitations of narrow genomic coverage and enhancing clinical utility.Consent Not applicable. This abstract reports only pooled, de-identified findings. No identifiable or sensitive patient information is included. Written informed consent was not requiredEthics Approval This study used biobank samples from Discovery Life Sciences and previously collected research specimens from registry studies. Ethics approval was obtained from Samsung Medical Center (IRB No. 2022-05-050) and from Seoul National University Hospital (IRB No. C-1111-102-387. All participants provided written informed consent in accordance with the Declaration of Helsinki.