Rapid detection and quantification of Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) in rainbow trout, are crucial to disease surveillance and encompass an essential component of BCWD research. Real-time, or quantitative polymerase chain reaction (qPCR) assays that have previously targeted the 16S rRNA gene of F. psychrophilum are complicated by polymorphisms and off-target amplification. Insignia primer and probe development software were used to identify a conserved single-copy signature sequence in the F. psychrophilum genome that codes for a hypothetical protein with unknown function. Primer and probes were used in a TaqMan qPCR assay that amplified 210 F. psychrophilum isolates with a lower limit of linear detection at 3.1 genome equivalents per reaction, with no amplification of 23 nontarget bacterial isolates. The assay was not inhibited by host spleen DNA or spleen homogenate. Methods were successfully applied to detect F. psychrophilum in rainbow trout from naturally occurring BCWD outbreaks and to quantify bacterial loads in experimentally infected rainbow trout. This assay will be applied to future studies to characterize disease pathogenesis in fish selectively bred for BCWD resistance.