Vanilla (Vanilla planifolia) is a crop of great biocultural importance in the Totonacapan region in Mexico; however, producers face various phytosanitary problems that decrease production. The main one being crop loss caused by the fungus, Fusarium oxysporum f. sp. vanillae, a pathogen that causes a disease whose symptomatology is root rot that in severe cases causes the death of plants. Therefore, the study of the pathogen is crucial, due to the scarcity of genetic variability of vanilla, a condition that makes the crops equally susceptible to the pathogen. The present work aimed to generate and molecularly and pathogenically characterize a fu-ngal isolates collection obtained from V. planifolia with root rot. Roots with rot were collected in six municipalities of the Totonacapan region, for fungal isolation, a semiselective culture medium was used, later, the molecular characterization was carried out through the amplification and sequencing of the partial region of the translational elongation factor 1α gene (TEF1). The pathogenic characterization was carried out on vanilla leaves in a humid chamber, inoculating medium discs with mycelium, and making observations for 18 days every 48 h. As results, 94 strains were obtained, of which 81 belong to the genus Fusarium (42 F. oxysporum strains), eight to Colletotrichum, three to Trichoderma and two it was not possible to identify them, only 23 Fusarium oxysporum strains showed to be pathogenic on vanilla leaves with different degrees of virulence. The results of the molecular and pathogenic characterization will be crucial for further studies of comparative genomics of Fusarium oxysporum.