Abstract In recent years, the advent of massively parallel “next-generation” sequencing technologies (NGS) has enabled substantial advances in the study of cancer. In combination with targeted DNA enrichment methods, high sequence coverage can be obtained for many different genes simultaneously, creating unique opportunities for clinical cancer diagnostics. However, the process of formalin fixation and paraffin embedding (FFPE) of tissue samples results in DNA-fragmentation by cross-linking to other biomolecules, posing a number of technical challenges for downstream mutation analyses. In this study, we have evaluated the HaloPlex target enrichment system for somatic mutation detection in DNA-extracts prepared from surgical non-small cell lung cancer (NSCLC) specimens with available paired tumor and normal tissue in FFPE blocks and matched fresh-frozen tumor and normal samples. In the design of the 56 gene panel, covering the most common mutations in lung colon cancer, several modifications to the standard method were introduced, including a reduced target fragment length, molecular barcoding, and “two strand capturing. We show that FFPE material can be used for HaloPlex based target enrichment and NGS using as little as 50 ng of DNA, i.e. compatible with yields readily available from small biopsies. By specifically capturing both strands for each target fragment, we were able to reduce the number of false positive errors due to FFPE induced artifacts by a factor of 4. Furthermore, the combined approach of molecular barcoding and two strand capturing allowed us to identify two low frequency variants, demonstrating a sensitive and accurate detection of somatic mutations. We believe that the HaloPlex method, provided some modifications are made, will be broadly applicable as a tool for somatic mutation detection. Genetic profiling of NSCLC FFPE tissues will add a new dimension to thoracic tumor pathology and will open vast opportunities for research as large, population-based cohorts of advanced stage NSCLC will be available for comprehensive molecular characterization. Citation Format: Johan Botling, Lotte N.J. Moens, Elin Falk-Sorqvist, Linnea LaFleur, Magnus Sundstrom, Patrick Micke, Mats Nilsson. Selector (HaloPlex) targeted resequencing of formalin-fixed paraffin-embedded (FFPE) specimens for mutation diagnostics in NSCLC. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A09.