The authors reported the isolation and genetic characterization of Sporothrix schenckii strains from natural environmental samples and commercial amended and garden soils. Twenty-six isolates were recovered and identified as S. schenckii by using both phenotypic and molecular methods. The majority of the strains were isolated from commercial amended and garden soils, indicating that these products represent an important reservoir of the fungus. Sequencing and phylogenetic analysis of the D1-D2 region of the 28S rRNA gene of environmental isolates, including S. schenckii ATCC 10268 and two Italian clinical strains, revealed a degree of difference sufficient to justify the separation of the examined isolates in two principal groups (environmental and clinical). Such separation in two groups is further supported by two well-conserved nucleotide polymorphisms, caused by single-base transitions, in the D1-D2 domain of rDNA. In this study, the presence in nature and/or in commercial products of S. schenckii is discussed. To our knowledge, this is the first study that reports the environmental isolation of S. schenckii from southern Italy.