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Ceratocystis wilt caused by Ceratocystis fimbriata is currently one of the most important diseases affecting Eucalyptus in Brazil. This disease is controlled by planting resistant clones; however, possible variability in the pathogen population may compromise the selection of resistant genotypes. Therefore, this study aimed to evaluate the aggressiveness of C. fimbriata isolates obtained from Eucalyptus spp, as well as their cultural characteristics and genetic variation of their ITS rDNA gene region. We found a significant isolate × clone interaction, with the isolate RM35 being the most aggressive and presenting a broader spectrum of aggressiveness, causing greater xylem discoloration on a larger number of clones. This isolate is the most suitable for artificial inoculations focusing on the selection of resistant materials. Clones CLR-236 and CLR-212 were identified as the most resistant and clones CLR-223 and CLR-240 as the most susceptible and those that are recommended as reliable comparators in artificial inoculations. All isolates were morphologically similar and differed from C. fimbriata from sweet potato by the formation of a wide mouth endoconidiophore that produces doliform endoconidia. According to the culture media and temperature applied, the most favourable conditions for mycelial growth were observed using malt extract agar (MEA) and temperatures ranging from 24 to 26°C. There was no correlation between sporulation and aggressiveness. Great variation in ITS sequences was observed, and a total of five ITS genotypes were identified among the ten isolates tested. [ABSTRACT FROM AUTHOR]