Tropomyosin receptor kinase (TRK) inhibitors have been approved for metastatic solid tumors harboringNTRKfusions, but the detection ofNTRKfusions is challenging. International guidelines recommend pan-TRK immunohistochemistry (IHC) screening followed by next generation sequencing (NGS) in tumor types with low prevalence ofNTRKfusions, including metastatic colorectal cancer (mCRC). RNA-based NGS is preferred, but is expensive, time-consuming, and extracting good-quality RNA from FFPE tissue is challenging. Alternatives in daily clinical practice are warranted. We assessed the diagnostic performance of RNA-NGS, FFPE-targeted locus capture (FFPE-TLC), fluorescence in situ hybridization (FISH), and the 5′/3′ imbalance quantitative RT-PCR (qRT-PCR) after IHC screening in 268 patients with microsatellite-instability-high mCRC, the subgroup in whichNTRKfusions are most prevalent (1–5%). A consensus result was determined after review of all assay results. In 16 IHC positive tumors, 10NTRKfusions were detected. In 33 IHC negative samples, no additional transcribedNTRKfusions were found, underscoring the high sensitivity of IHC. Sensitivity of RNA-NGS, FFPE-TLC, FISH, and qRT-PCR was 90%, 90%, 78%, and 100%, respectively. Specificity was 100% for all assays. Robustness, defined as the percentage of samples that provided an interpretable result in the first run, was 100% for FFPE-TLC, yet more limited for RNA-NGS (85%), FISH (70%), and qRT-PCR (70%). Overall, we do not recommend FISH for the detection ofNTRKfusions in mCRC due to its low sensitivity and limited robustness. We conclude that RNA-NGS, FFPE-TLC, and qRT-PCR are appropriate assays forNTRKfusion detection, after enrichment with pan-TRK IHC, in routine clinical practice.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00428-023-03538-1.