Abstract: We used RAPD (random amplification of polymorphic DNA) to analyze the phenotypic and genotypic characteristics of Sporothrix schenckii isolated from five geographic regions of Brazil, from clinical, animal, and environmental sources. Our results yielded a significant difference (P<0.01) in the mean conidial area of S. schenckii animal isolates (2.96 ± 1.07) compared with those of clinical isolates (fixed form, 2.33 ± 0.53; lymphocutaneous form, 2.37 ± 0.43). There was no association among S. schenckii clinical isolates and geographic region. Isolates from the Northeast region exhibited the lowest thermotolerance (% growth inhibition) at 35ºC ( x = 49.23% ± 17.25) and at 37ºC (70.43% ± 10.93%). Northern isolates exhibited the highest thermotolerance at 35ºC (12.82% ± 5.73%) and at 37ºC (23.81% ± 8.27%). RAPD with a 10-mer primer OPD-18 generated 67 PCR fingerprint patterns. The 151 S. schenckii isolates fell into seven major clusters with such great genetic diversity that an association of isolates with clinical forms or geographic areas could not be determined, even with investigations focused on more restricted geographic areas. The main physiological characteristics of Brazilian S. schenckii isolates were also characterized, including osmophilia, halophilia, pH tolerance, urease activity, casein hydrolysis, and gelatinase, proteinase, and DNAase production.