Abstract Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, whole-genome sequencing (WGS) of these samples has remained challenging due to the difficult process of isolating high-quality DNA and distinguishing true variant calls from artifacts. As evidence of these challenges, only four WGS studies of FFPE samples have been published to date. Previous work has attempted to overcome these challenges by using whole-exome sequencing or smaller exon panels at high coverage depths with publications reporting success from less than half to most samples. Thus, there has not been widespread adoption of using FFPE samples for next-generation sequencing. We developed a novel DNA extraction and library prep protocol for whole-genome sequencing of FFPE tissue samples. This method produces larger DNA fragments that can be successfully used for WGS. We previously presented results of a large esophageal cancer cohort where we sequenced over 1,000 samples in tumor-normal pairs with data equivalent to that generated from fresh-frozen tissues. We recently expanded the tumor types, ages, and sources and achieved similar results. We have assayed FFPE tissues derived from breast cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma. These samples were sourced from several different labs, which have used variations on the standard fixation protocol. Some of these FFPE samples were over 15 years old. Variant calling provides excellent results without excessive C>T mutations. The coverage uniformity was very good, and CNV calls were made with results comparable to published TCGA data. These samples have been analyzed for MSI and TMB, and the results are comparable to those using fresh tissues. Our method makes new types of clinical studies possible utilizing FFPE samples that have been generated as part of standard hospital procedures. This method also facilitates performing studies comparing primary and recurrent cancers easier to establish and run as recurrent cancer cases can be enrolled and stored FFPE primary samples used to establish a cancer baseline. Citation Format: Shannon T. Bailey, James Lund, Muhammad Ekram, Wanfeng Yu, Richard T. Williams, Hongye Sun, Jeffrey R. Gulcher. High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1706.