Introduction: Gene fusions, usually the result of chromosomal rearrangements, are frequently associated with many cancer types, and hence clinically actionable, making fusion detection an important part of cancer disease management. We developed a new, optimized version of the Seraseq® Fusion RNA Reference Material, and demonstrate its consistent performance across different NGS enrichment assays, sequencing coverage depths, bioinformatics pipelines, and RNA mass inputs from a multi-lab investigation (Sites A, B, C, D & E). Methods: Biosynthetic DNA was used for transcription of 18 RNA fusions, including more common fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. The in vitro transcribed RNAs were mixed with total RNA extracted from GM24385 reference cell line (The 1000 Genomes Project, Coriell). Digital PCR with TaqMan® chemistry was used to determine the target fusion RNA concentration and serve as the “truth” data set for comparison to NGS, which can be variable depending on input, assay, and bioinformatics. The fusion-total RNA mix was analyzed by five external laboratories; it was tested using the ArcherDx FusionPlex™ Solid Tumor Panel (Site A, Site B, Site C), the ArcherDx FusionPlex™ CTL Panel (Site C), the ArcherDx FusionPlex™ Lung Panel (Site B), a custom ArcherDx FusionPlex™ Panel (Site D), and the TruSight Tumor 170 Panel (Site E). Results: All eighteen (18) fusions in the new Seraseq Fusion RNA Mix v4 reference standard were detected as expected on each NGS platform with an average of greater than 85% of on-target reads across all assays. Even at inputs as low as 20 ng, all 18 fusions were typically detected above fusion-calling thresholds. In general, the results for individual fusions among the different NGS panels and among replicates were concordant, with observed variance in reads across some fusion junctions between assays and replicates. Within FusionPlex assay results, the average percent of reads supporting the fusion call across all fusions was about 63%, regardless of input (a range between 20 to 250 ng). Collectively, the multi-lab results confirm that the Seraseq® Fusion RNA Mix v4 reference standard is compatible with both amplicon and hybridization-capture based NGS assays. Conclusions: Seraseq RNA Fusion Mix v4 has broad NGS assay compatibility and allows for reliable and simultaneous detection of 18 clinically relevant RNA fusions even at low input amounts. The data from a multi-lab study support the use of this reference standard for targeted NGS assay development, assay validation, bioinformatics pipeline optimization, and as positive controls in clinical NGS RNA fusion assays. The biosynthetic manufacturing approach produces reference materials that provide consistent results for a wide variety of common and rare gene fusions. Citation Format: Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella. Consistent performance of highly multiplexed RNA fusion reference materials across different NGS assays in a multi-lab study [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 1709.