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To identify specific sequences in the fish pathogen Flavobacterium columnare not shared by Flavobacterium johnsoniae.Suppressive subtractive hybridization (SSH) was used to selectively amplify and clone F. columnare-specific sequences. A highly virulent strain of F. columnare was used as tester and the type strain of F. johnsoniae was used as driver. After library construction, 192 clones were selected and sequenced. From those, 110 clones contained unique F. columnare-specific sequences that were verified using dot blot hybridization. Sequence sizes ranged from 55 to 872 bp with 45 363 bp sequenced in total.Specific F. columnare sequences representing all but one (motility related) functional categories were annotated. Several putative virulence factors were identified in F. columnare such as a collagenase, a chondroitinase, proteases, as well as drug resistance and iron transport-related genes.Suppressive subtractive hybridization is a cost-effective method for identifying genetic differences between Flavobacterium spp. The number of sequences available from F. columnare has been doubled.