Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into three genotypes. The isolates within the genotypes were further grouped based on RFLP of the 16S-23S rDNA spacer. The first genotype had five strains that were further divided into group A (4 strains) and B (1 strain) while the second genotype had 10 strains that were also further divided into group A (4 strains) and B (6 stains). The third genotype had 12 isolates with no differences in the RFLP patterns of the 16S-23S rDNA spacers. The 16S rRNA gene sequences representing the three identified genotypes were compared to the different published sequences by phylogenetic analysis and the results showed the American genotypes 1, 2 and 3 corresponding to genomovar 1, 2, and 3, respectively, reported by Triyanto and Wakabayashi [Triyanto, Wakabayashi H. Genotyping of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 1999; 34: 65-71]. The study demonstrates a method for RFLP and sequencing of the 16S rRNA gene and the 16-23S rDNA spacer as a useful tool in epidemiological studies of F. columnare.