Soybean (Glycine max L.) is the most widely cultivated crop in the world and an important commodity. Besides its main role in human nutrition, this grain is also used in animal feed and production of biofuels, among other purposes. Due to these factors, soybean became important in the global economy and is the most exported agricultural product from Brazil. Fungal diseases are among the limiting factors on soybean production; sudden death syndrome, caused by Fusarium spp., has been responsible for severe losses on this crop in Brazil. Four species of Fusarium can be considered causal pathogens: F. brasiliense, F. crassistipitatum, F. tucumaniae, and F. virguliforme. The Fusarium spp. isolates characterized in this work were collected in different soybean-producing regions in Brazil. The genetic variability of these isolates was determined through the random amplified polymorphic DNA (RAPD) technique. Disease severity was evaluated on moderately resistant and susceptible soybean cultivars in greenhouse trials. RAPD analysis demonstrated a great genetic diversity among the isolates and a clear tendency to split into two main species groups, F. tucumaniae and F. brasiliense, both prevalent in Brazil. The disease severity experiments, in which soybean plants were artificially inoculated, have shown that all isolates caused significant damage to the seedling root system. In fact, the genetic diversity of isolates does not correlate with disease severity, and also does not correlate with geographic distribution.