In this paper, we report a patient in whom Mycobacterium marinum sporotrichoid infection was diagnosed using polymerase chain reaction (PCR) amplification of the 16S rRNA gene and subsequent analysis of the amplified product in a reverse cross-blot hybridization assay with mycobacterial species-specific probes. This molecular method allowed us rapidly to detect and identify this organism directly in the patient's lesional skin biopsy rather than in cultures in conventional media. The identification provided by PCR-reverse cross-blot hybridization assay was confirmed by examination of the morphological and biochemical features and by high-performance liquid chromatography analysis of mycolic acid from the clinical isolate, suggesting the validity of our molecular approach.