Abstract Clinical detection of genomic rearrangements currently depends on a range of complementary and overlapping methods including fluorescence in situ hybridization (FISH), karyotyping, and DNA and/or RNA sequencing. This requirement for multiple tests results in higher total costs, extended analysis time, and increased usage of sometimes limited tumor material. In our laboratory, we apply long high-fidelity reads to human samples using PacBio circular consensus sequencing (HiFi-GS) of 12-18kb DNA fragments, which offers better structural variant detection relative to short-read sequencing. Here, we assess the ability of HiFi-GS to detect clinically relevant genomic rearrangements (CRGRs) in pediatric leukemia. We selected 11 pediatric leukemia cases with corresponding normal (remission) samples, including 7 cases of B-cell acute lymphoblastic leukemia (B-ALL) and 4 cases of acute myeloid leukemia (AML); 5 of these 11 leukemia cases had known CRGRs as below. The remaining 6 leukemia cases had also undergone clinical genetic profiling via karyotyping, FISH, microarray, and sequencing yet a clinical grade genomic driver was not identified. DNA from each sample was sequenced on a Revio instrument (PacBio, Menlo Park, CA) to a target depth of 30x. The PacBio Human Whole Genome Sequencing (WGS) workflow was used to process HiFi reads for haplotagged alignment and phasing, followed by somatic structural variant calling using Severus. Subsequent analysis focused on the presence of break ends in known cancer-relevant genes. We found that HiFi-GS detected the known CRGRs in all 5 cases with prior findings, providing precise break ends and clarifying sometimes unclear cytogenetic observations (such as exact partner genes), in these cases. These included detection of an ETV6-RUNX1 fusion, NUP98-NSD1 fusion, KMT2A-AFF1 fusion, KMT2A-AFDN fusion, and RUNX1-RUNX1T1 fusion. HiFi sequencing also detected CRGRs in 2 of the 6 “undiagnosed cases”; namely, one case of ZNF384-TCF3 fusion and one case of KMT2A-MLLT10 fusion. The former can be cytogenetically cryptic, while the latter can often be missed due to its potentially complex nature. Our results demonstrate that HiFi-GS can reliably detect CRGRs, bringing us closer to the promise of a single comprehensive genetic test for cancer characterization. Additional work examining other somatic structural and copy number variants (i.e., inversions, deletions, and duplications) in these cases, as well as oncogenic sequence variants, is ongoing. Citation Format: Byunggil Yoo, Ayse Keskus, Chengpeng Bi, Lisa Lansdon, Tanveer Ahmad, Irina Pushel, Adam Walter, Margaret Gibson, Erin Guest, Tomi Pastinen, Mikhail Kolmogorov, Midhat S. Farooqi. Long-read sequencing of pediatric leukemia identifies clinically relevant genomic rearrangements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1763.