Fusarium wilt is considered as one of the most important diseases that affects tomato (Solanum lycopersicum L.) cultivation. The objective of this study was to identify and characterize Fusarium species with the potential to cause tomato wilt using morphological and molecular approaches, in order to generate the necessary information to achieve effective control of this disease. Fusarium isolates were found associated with commercial grown cultivars with disease incidence ranging from 10 to 85%. Forty isolates were identified by morphological characteristics as Fusarium oxysporum (38) and as Fusarium sp. (2). The isolates were evaluated for their pathogenicity on healthy tomato seedlings, which presented root rot at 20–35 days after inoculation. Fifteen of the most pathogenic isolates were analyzed with the internal transcribed spacer (ITS) region of DNA and the partial sequence of the translation elongation factor 1α (EF-1α). Isolates associated with tomato wilt, were identified molecularly as Fusarium oxysporum (13), Fusarium circinatum (1), and Fusarium andiyazi (1). Both analysis revealed that the mayor agent of tomato wilt in Mexico was F. oxysporum. This finding provides relevant information on tomato wilt in Mexico to decide the proper control methods for the pathogen.