ABSTRACT Renibacterium salmoninarum , a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2 . We found evidence for a third msa gene, msa3 , which appears to be a duplication of msa1 . Unlike msa1 and msa2 , msa3 is not present in all isolates of R. salmoninarum . The presence of the msa3 locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the msa3 locus reduced median survival in juvenile chinook salmon ( Oncorhynchus tshawytscha ) by an average of 34% at doses of ≤10 ⁵ cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 10 ⁶ cells per fish. The phenotype associated with the msa3 locus and its nonuniform distribution may contribute to observed differences in virulence among R. salmoninarum isolates.