Abstract A typical cancer somatic mutation identification workflow involves using a standard human reference genome for aligning the sequencing reads and annotating the uncovered somatic variants. The completeness and the correctness of the reference genome, and the haplotype representations may all impact the sequencing read alignments and subsequent somatic variant identifications. However, the exact impacts of the underlying references being used on somatic mutation analysis has not been investigated. In this study, we selected 3 human reference genomes, including GRCh38, T2TCHM13, and a personalized genome (PG) that was derived from a normal cell line, and 12 tumor normal paired replicates of Illumina short read sequencing data for the HCC1395 breast cancer cell line and a matched normal cell line, and aim to illustrate and quantify the effects of the reference genomes on the accuracy of somatic SNV/SV mutation detection using various somatic variant callers. Our analysis shows that the use of a personalized haplotype-specific genome assembly, rather than an unrelated genome such as GRCh38 or T2TCHM13 as a reference, not only improves read alignments for short-read sequencing data but also ameliorates the detection accuracy of somatic SNVs and SVs. While most somatic mutation calls are identical or equivalent, we identify GRCh38 and T2TCHM13 specific somatic mutations that may need to be avoided for further pursuit of incorrect treatment options. We uncover novel somatic mutations only when personalized genome assembly is used as a reference, some of which overlap with genes involving with pathways related to cancer invasion and metastasis (e.g., CDH23, ST14 etc.). The predicted somatic SVs are found more precise. Such results may provide additional interventional target choices for patients, researchers or clinicians, and physicians to look into further for personalized patient care. Citation Format: Chunlin Xiao, Valerie Schneider. Assessment of human reference genomes on cancer somatic mutation detection in tumor-normal paired reference samples using whole genome short-read sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2033.