Simple SummaryThe identification of potential molecular alterations is standard in the diagnostic pathway of non-small-cell lung cancer (NSCLC). The aim of this study is to determine the prevalence ofNTRKfusions in NSCLC in a routine diagnostic setting using immunohistochemistry, fluorescence in situ hybridization, and RNA-based next-generation sequencing. A total of 1068 unselected consecutive patients with NSCLC were screened in two scenarios, either with initial IHC followed by RNA-NGS (n= 973) or direct FISH testing (n= 95). In total, 0.2% of all patients wereNTRKpositive. Both RNA-NGS and FISH are suitable to determine clinically relevantNTRKfusions in a real-world setting. RNA-NGS or FISHNTRKpositive results were mutually exclusive with alterations inEGFR/ALK/ROS1/BRAF/RETorKRAS.Abstract(1) Background: The main objectives of our study are (i) to determine the prevalence ofNTRK(neurotrophic tyrosine kinase) fusions in a routine diagnostic setting in NSCLC (non-small cell lung cancer) and (ii) to investigate the feasibility of screening approaches including immunohistochemistry (IHC) as a first-line test accompanied by fluorescence in situ hybridization (FISH) and RNA-(ribonucleic acid-)based next-generation sequencing (RNA-NGS). (2) Methods: A total of 1068 unselected consecutive patients with NSCLC were screened in two scenarios, either with initial IHC followed by RNA-NGS (n= 973) or direct FISH testing (n= 95). (3) Results: One hundred and thirty-three patients (14.8%) were IHC positive; consecutive RNA-NGS testing revealed two patients (0.2%) withNTRKfusions (NTRK1-EPS15(epidermal growth factor receptor pathway substrate 15) andNTRK1-SQSTM1(sequestosome 1)). Positive RNA-NGS was confirmed by FISH, andNTRK-positive patients benefited from targeted treatment. All patients with direct FISH testing were negative. RNA-NGS- or FISH-positive results were mutually exclusive with alterations inEGFR(epidermal growth factor receptor),ALK(anaplastic lymphoma kinase),ROS1(ROS proto-oncogene 1),BRAF(proto-oncogene B-Raf),RET(rearranged during transfection) orKRAS(kirsten rat sarcoma viral oncogene). Excluding patients with one of these alterations raised the prevalence ofNTRK-fusion positivity among panTrk-(tropomyosin receptor kinase-) IHC positive samples to 30.5%. (4) Conclusions:NTRKfusion-positive lung cancers are exceedingly rare and account for less than 1% of patients in unselected all-comer populations. Both RNA-NGS and FISH are suitable to determine clinically relevantNTRKfusions in a real-world setting. We suggest including panTrk-IHC in a diagnostic workflow followed by RNA-NGS. Excluding patients with concurrent molecular alterations toEGFR/ALK/ROS1/BRAF/RETorKRASmight narrow the target population.