In this study, the mechanism conferring multiple drug resistance in several strains of flavobacteria isolated from the ovarian fluids of hatchery reared 3-year old brook trout Salvelinus fontinalis was investigated. Metabolic fingerprinting and 16S rRNA gene sequences identified the isolates as Flavobacterium johnsoniae. The isolates exhibited multiple resistances to a wide range of antimicrobial classes including penicillin, cephem, monobactam, aminoglycoside, and phenicol. Although plasmids and other transposable elements containing antimicrobial resistance genes were not detected, the isolates did contain a genomic sequence for a chloramphenicol-inducible resistance-nodulation-division family multidrug efflux pump system. Efflux pumps are non-specific multidrug efflux systems. They are also a component of cell-cell communication systems, and respond specifically to cell membrane stressors such as oxidative or nitrosative stress. Understanding of efflux pump mediated antibiotic resistances will affect efficacy of clinical treatments of fishes associated with F. johnsoniae epizootics.