Potted plants of Mammillaria painteri (Cactaceae) showing symptoms of stem rot were collected from a nursery in Imperia province, Liguria region, Italy. Isolations from internal rotting tissues allowed gave constantly similar fungal colonies. Morphological characteristics of the isolates identified them as Fusarium oxysporum. Molecular analyses of the elongation factor 1α (EF1α) and RPB2 genes confirmed the identification. Analysis of part of the intergenic spacer (IGS) region of the ribosomal DNA identified the pathogen as F. oxysporum f. sp. opuntiarum. In pathogenicity tests, stems of M. painteri plants were inoculated with representative F. oxysporum f. sp. opuntiarum isolates. Approx. 30 d after the inoculation, yellowing appeared around the inoculated wounds. The inoculated stems then rotted developing symptoms similar to those observed in greenhouse-grown plants. This is the first report of F. oxysporum f. sp. opuntiarum on M. painteri.