The cultivation of yerba-mate (Ilex paraguariensis) has been intensified in the State of Rio Grande do Sul, as result of the valuation of the price paid to the producer. However, studies related to Fusarium pathosystem vs. yerba-mate are still insufficient, so that many producers have faced significant losses in herbal production due to the occurrence of the roots rotting. Considering the above, the objectives of this study were: a) morphologically characterize the Fusarium spp. isolates collected in sick plants of yerba-mate; b) select efficient morphological characters on separating the Fusarium spp. isolates in similarity groups; c) identify the Fusarium spp. isolates to species level through the sequencing of the genomic region of elongation factor 1-α (TEF-1α); and d) testing the pathogenicity of the Fusarium spp. isolates selected based on the morphological characterization. For such, collections for the pathogen isolates were made in five cities and the collection points were georeferenced. Thirty-nine isolates were morphologically characterized by variables as mycelial growth, sporulation, macroconidia length and width, microconid format, colonies pigmentation and formation of chlamydospore, and tested for pathogenicity by substrate inoculation. The variables used in the morphological characterization were efficient in discriminating the isolates in seven groups, especially length of macronidia associated to sporulation. Seven pathogenic isolates of Fusarium spp. were identified, also through molecular sequencing of the TEF-1α gene region. The sequencing of the TEF-1α region consolidated the identification of pathogenic isolates as F. solani and F. oxysporum.