Flavobacterium columnare is an important bacterial pathogen of fish with a wide genetic variability within the species. This intra-species diversity has been termed as genomovars and genomovar groups on the basis of Restriction Fragment Length Polymorphisms of 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR), respectively. In this study, we demonstrate the source of genetic heterogeneity in the F. columnare by sequence analysis of ISR. The length of ISR sequences of different genomovars varied from 553 to 592 nucleotides, while the similarity among sequences ranged from 76.1 to 92.6%. A common ISR structure with tRNA^Ala and tRNAIle embedded within the sequence was identified in all the genomovars of F. columnare. The results show that strains of F. columnare can be categorized into five genomovar groups based on the heterogeneity of the ISR sequences. Of these, strains belonging to Genomovar I and II can be sub-divided into two groups each; while strains of Genomovar III belong to one group. Sequence similarity between genomovar groups was lower for ISR (76.1-92.6%) as compared to 16S rDNA (96.1-99.4%) indicating its ability to resolve closely related groups within the genomovars of F. columnare. The main source of variation between the genomovar groups is the presence of three hyper variable regions (V1, V2, and V3) in the ISR. Of the three, V3 was found to be the most heterogeneous region and was found to be useful in assigning a genomovar group to an individual strain of F. columnare.