Restriction fragment length polymorphism (RFLP) in mitochondrial DNA (mtDNA) from 25 environmental isolates of Sporothrix schenckii from northeastern China was investigated. Based on the mtDNA-RELP patterns with Hae III, 6 isolates were confirmed to be S. schenckii, while the other 19 isolates were confirmed to be species distinct from S. schenckii. The mtDNA RFLP patterns of the 19 non-S. schenckii were identical to each other. The non-S. schenckii isolates could not be discriminated from S. schenckii by their macro- or micro-morphological features, and were not pathogenic in guinea pigs. Serological and delayed hypersensitivity cross-reactions were found between S. schenckii and the non-S. schenckii species, suggesting antigenic similarity. These results indicate that RFLP analysis of mtDNA is essential for the identification of environmental isolates of S. schenckii.