Flavobacterium columnare is a ubiquitous Gram-negative bacterium that causes columnaris disease in a wide variety of fish worldwide. Timely detection of this bacterium is important to prevent its spreading and to reduce the economic loss to fish farmers. We developed a TaqMan-based real-time polymerase chain reaction (PCR) targeting a 113 bp nucleotide region of the chondroitin AC lyase gene of F. columnare G4. Specificity of the assay evaluated with 20 isolates of F. columnare and 15 other taxonomically or ecologically related bacteria revealed that the primers and probe were 100% specific for detection of F. columnare. The sensitivity limit of detection of F. columnare in pure cultures, over a range of dilutions [3.1 × 100–3.1 × 106 colony-forming units (CFU) mL−1], was observed to be ∼3 bacterial cells. The lowest limit of detection in nucleic acids from pure culture of F. columnare was 5.4 fg and the assay was linear with the log of amount of nucleic acid (R2=0.994) over that range (5.4 ng–5.4 fg). In tissues (blood, gills and kidney) of F. columnare experimentally infected fish, the bacterial numbers measured by TaqMan real-time PCR ranged from 3.4 × 100 to 9.5 × 105 CFU mL−1. In both F. columnare experimentally infected and spiked samples, positive PCR results were confirmed by bacteriological culture with 100% agreement. The TaqMan real-time PCR developed in this study is specific, sensitive and reproducible for the detection and quantitation of F. columnare in infected fish.