Acacia koa (koa), a native tree in Hawaii, suffers from a dieback caused by Fusarium oxysporum. Pathogenicity tests, vegetative compatibility group (VCG) tests, and amplified fragment length polymorphisms (AFLP) analyses were conducted on Fusarium isolates recovered from diseased koa. Koa seedling mortality with individual strains ranged from 0 to 85%, with 42% of the strains killing no seedlings. Thus, strains of F. oxysporum recovered from dying koa trees may or may not be virulent. In addition to F. oxysporum, F. pseudocircinatum strains were isolated from diseased koa; however, they were either nonvirulent or had weak virulence. This is the first report of F. pseudocircinatum in Hawaii. The 46 strains of F. oxysporum and F. pseudocircinatum were grouped into 16 VCGs, but 86% of the highly virulent strains belonged to VCG 2. In AFLP analyses, strains from the same VCG generally clustered with one another. Identification of the same set of strains using VCG, AFLP, and pathogenicity tests showed that the highly virulent strains are genetically close and that high virulence toward koa is not a property of all strains of F. oxysporum. Thus, VCG 2 with the corresponding AFLP data is a significant biological entity for which we propose the name F. oxysporum f. sp. acaciae to reflect its virulence on koa.