AbstractRecent research has identified four distinct genetic groups among isolates ofFlavobacterium columnarethrough multilocus phylogenetic analyses; however, there are no quick methods to determine the genotype of an isolate. The objective of this research was to develop a multiplex PCR to rapidly genotypeF. columnareto genetic group. Comparative bacterial genomics was used to identify regions in the genomes unique to each genetic group, and primers were designed to specifically amplify different sized amplicons for each genetic group. The optimized assay was demonstrated to be specific for each genetic group andF. columnare, and no specific amplicons were generated using gDNA from a panel of otherFlavobacteriumspp. and bacterial fish pathogens. The analytical sensitivity of the assay ranged from 209 to 883 genome equivalents depending on the genetic group. The multiplex PCR was evaluated by genotyping a panel of 22 unknownF. columnareisolates and performing DNA sequencing of thednaKgene in parallel. The results demonstrated 100% accordance between multiplex PCR results and assignment to genetic group via phylogenetic analysis. The multiplex PCR provides a useful tool for assigning an unknown isolate to genetic group and may be used to determine which genetic groups ofF. columnareare circulating and most predominant in different aquaculture industries.