Abstract Long non-coding RNAs (lncRNAs) play important roles in all the central aspects of the gene expression cascade with wide ranging effects on cell proliferation and differentiation. As such, lncRNAs are highly promising additions to many cancer biomarkers portfolios and enable more precise diagnostic staging. This, in turn, provides a clearer prognosis and treatment options of different cancers. Direct visualization and quantification of lncRNAs in single cells and in tissue are thus emerging as essential tools for both basic and clinical research. Interestingly, studies have demonstrated that transcript levels are more strongly correlated to clinical traits than the corresponding protein levels. Because RNA biomarkers have the potential to contribute to the development of targeted cancer therapies, there is also an obvious need for a robust and dependable methodology to discover and validate these RNAs. RNA fluorescence in situ hybridization (RNA FISH) provides a powerful means to detect RNA biomarkers in single cells, while still maintaining tissue morphology. Significant advances in RNA FISH technology, such as the hybridization of pools of singly labeled fluorescent 20-mer oligonucleotides to the RNA target, now afford specific and sensitive multiplex detection of RNA to yield information on the RNA's distinct spatial distribution within cells and tissue. Data is presented on the design of probe sets against multiple disease relevant pre-lncRNAs and against the introns of their precursors, as well as on the simultaneous dual-channel imaging of the pre-lncRNAs and mature lncRNAs. Because the absolute majority of post-transcriptional processing, including splicing of mRNAs and lncRNAs, occurs co-transcriptionally, the location of introns can be used as a proxy nuclear location for the encoding gene. We find mature lncRNAs to locate at or close to the site of transcription, but also well away from it. Next, we present data on emerging non-coding RNA biomarkers in breast and prostate cancer tissue and in cell lines utilizing RNA FISH. In summary, the results and tools presented here will contribute to advancing the current capabilities of the detection and treatment of specific cancers, as well as in the continued discovery and development of cancer drug candidates. Citation Format: Arturo V. Orjalo, Jr., Hans E. Johansson. Stellaris® RNA fluorescence in situ hybridization (RNA FISH) for the detection of long non coding RNA biomarkers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-44.