Genus Flavobacterium contains species that are important pathogens of fish impacting aquaculture worldwide, causing economic loss, outbreaks in fish farms occur in wide range of temperatures. This genus is considered cosmopolitan in freshwater habitats, until now there are 312 species assigned to the genus, according to taxonomy database NCBI. A lot of virulence related characteristics and distributions of species were evidenced, but the influence of temperature in expression of virulence traits is not well understood. So, the aim of this report is understood if 10 Flavobacterium-Chyseobacterium strains isolated from clinical cases in native Brazilian fishes, tilapias and 1 water-source strain belonging to the species, Flavobacterium davisii, F. oreochromis, F. indicum, F. inkyongense, unknown Flavobacterium sp. and Chyseobacterium gambriani have risk factor associated with temperature. The characteristics, production of casein proteolytic enzymes and cultivation in stable culture without shaking were quantified in three different temperatures 22, 25 and 28ºC. First strains were cultured in G Agar media for 24 hours and colonies were suspended in 7,5 mL of sterile G broth with 2x NaCl to avoid clump formation and ensure an well homogenized inoculum. After the incubation, 100uL of each overnight culture were passed to a new 7,5 mL sterile G broth, all the culture conditions were done in triplicate. To access the proteolysis quantification, the bottom of 200uL tips was used to form a 6mm diameter hole in the G Agar supplemented with 2% (v/v) of skimmed milk. The bacterial cultures at different temperatures were used to inoculate modified plates. After 6 hours of incubation in the aforementioned temperatures, a clear halo appears, and this halo was measured (mm) to compare the production of casein-hydrolytic enzymes in different incubation conditions. The proteolysis assay demonstrated different profiles of hydrolyze by different strains (n=6) of F. oreochromis, 4/6 produced more proteolytic enzymes at 28ºC, 1/6 produced equal proportion of this virulent tract both in 25 and 28ºC but lower at 22 ºC and 1 strain have higher production of this kind of enzymes at 25 ºC supposing heterogenity of expression in this trait by different strains of F. oreochromis. The optimal temperature to casein proteolysis of other 3 strains of species C. gambiriani, F. inkyongense and unknown Flavobacterium sp. was 28, 25-28 and 25 ºC respectively. No reliable results were attributed to F. davisii and F. indicum species using this protocol with 6 hours of incubation, only one strain of each species was evaluated. We hope that these results will help to investigation of phenotypically expression of proteolytic activity by Flavobacterium spp. and the application of this method with different proteins sources, we wait improving the knowledge of Flavobacterium pathogens and the mitigation of damage caused by these agents in fish farms.