Two pathogenic special forms (f. sp.) of the Fusarium oxysporum species complex f. sp. lycopersici (Fol) and f. sp. radicis-lycopersici (Forl) are morphologically indistinguishable. Although they are pathogenic to the same host genus Lycopersicon (tomato), and infect the same tomato cultivar, they form distinct diseases; Fol causes wilt and Forl causes crown rot and root rot. These two special forms apparently exist as genetically isolated populations, based on vegetative compatibility and molecular variation at the DNA level. In seeking efficient diagnostic tools for differentiating Fol and Forl isolates, we examined three techniques: isozyme analysis, mitochondrial DNA (mtDNA) RFLP by HaeIII-digestion of total genomic DNA, and an osmotic method using high performance liquid chromatography (HPLC) to detect fungal pigments. The isolates were collected from geographically widespread locations. Distinct HPLC-profile differences were found between an endophytic non-pathogenic isolate and the other pathogenic isolates. However, the direct mtDNA RFLP technique proved to be an efficient diagnostic tool for routine differentiation of Fol and Forl isolates.