Aims: To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare Methods and Results: Genetic variability among Fl. columnare isolates was characterized using restriction fragment length polymorphism analysis of the 16S rDNA gene, intergenic spacer region (ISR) sequencing, and amplified fragment length polymorphism (AFLP(R)) fingerprinting. Thirty Fl. columnare cultures isolated from different fish species and geographical origins as well as reference strains were included in the study. Fifteen isolates belonged to genomovar I while eleven were ascribed to genomovar II. Analysis of the ISR sequence confirmed the genetic differences between both genomovars but revealed a higher diversity among genomovar I isolates. The maximum resolution was provided by AFLP(R) fingerprinting, as up to 22 AFLP profiles could be defined within the species. Conclusions: We confirmed the division of Fl. columnare isolates from cultured fish into different genogroups. We showed that both genomovars I and II are present in channel catfish from the US. We described a unique genetic group represented by four Fl. columnare isolates from tilapia in Brazil which appears to be related to both genomovars. We were able to further subdivide the species by analysing the ISR. Finally, the use of AFLP(R) allowed us to fingerprint the species at clone level without losing the higher genetic hierarchy of genomovar division. Significance and Impact of the Study: This paper reports on an extensive assessment of the use of molecular tools for the study of the epidemiology of the fish pathogen Fl. columnare.