Ninety-seven motile Aeromonas strains were isolated over a period of a year from samples of water and sediment collected at different sites along a river. Strains were regularly recovered from all samples, regardless of the source of isolation or seasonal conditions. Isolates were biochemically characterized by the API 20NE system (Analytab Products, Plainview, N.Y.) and classified as Aeromonas hydrophila (74 strains), Aeromonas sobria (11 strains), and Aeromonas caviae (12 strains). Despite the high level of homogeneity observed in their biochemical patterns, they displayed different degrees of virulence for fish; 72.02% of A. hydrophila isolates and 63% of A. sobria isolates were virulent for fish by intramuscular challenge, but lower frequencies of virulence were observed when intraperitoneal injections were used. All A. caviae strains proved to be avirulent. Caseinases, hemolysins, and Vero cytotoxins were produced by 100, 91, and 94.59%, respectively, of A. hydrophila strains and with lower frequencies and lower caseinase activities by A. sobria isolates. No correlation was found between these activities and the degree of virulence of the strains for fish. Most hydrophobic strains seem to be concentrated in A. caviae, A. sobria, and avirulent A. hydrophila groups. Known virulence markers commonly associated with virulent strains (acriflavine negative and self-pelleting negative and precipitation after boiling positive phenotypes) had a low representation in the total of strains studied and were not associated with virulence.