Fusion genes are the result of genomic structural variants that arise when the coding region of two previously independent genes become joined together and can be a major driving cause of cancer development. Many gene fusion events have been classified as clinically-relevant and are targets for both diagnostic applications, such as TMPRSS2-ERG in prostate cancer, and therapeutic applications, such as CD74-ROS1 and EML4-ALK in non-small cell lung cancer. Given the potential clinical benefits of early detection, there is significant interest in developing highly sensitive and accurate diagnostic assays to detect cancer using RNA transcripts of these gene fusions as biomarkers. A significant barrier to the development of these diagnostic assays is the lack of a reliable and renewable gene fusion positive control for use in assay development and analytical validation.   Here, we describe the development and performance of a new fusion reference material: the Twist Pan-cancer RNA Fusion Control, a highly-multiplexed positive control designed to be spiked into a selected RNA background or as a stand-alone positive control. The Twist Pan-cancer RNA Fusion Control provides a wide selection of 80+ common and curated cancer targets for analytical validation and assay development. The synthetic RNAs are designed centered around the known and documented fusion sites with 750 nt of RNA 5′- of and 3′- of the fusion junction and is suitable for both short read sequencing and qPCR experiments. Pooled synthetic fusion RNAs are quantified, pooled and normalized to similar molar ratios, analyzed for uniformity by NGS, and ddPCR’d to establish a highly precise pool concentration. Finally, in fusion detection NGS experiments, we observe over a 90% recall rate of fusion events, compared to a 100% recall rate during QC, highlighting the contribution of panel and bioinformatic approach to detection and the importance of controls.   The Pan-cancer RNA Fusion Control is designed to serve as a spike-in or stand-alone positive control and specifically pairs well with a clinically-relevant target enrichment (TE) panel within the Twist Targeted Enrichment for Gene Expression solution. This control sample positive for RNA fusions can be applied in both qPCR and NGS workflows to validate panel/probe sets, establish limits of detection, and monitor ongoing assay performance. The Twist Pan-cancer RNA Fusion Control provides a valuable one-stop solution to assay developers seeking reference materials for a wide array of cancer-associated fusion transcripts.