Two fatty acid analysis protocols (the MIDI and a modified MIDI method) were investigated for their utility to characterize and differentiate Rhizoctonia oryzae and R. oryzae-sativae isolates from four countries. Only the modified MIDI method permitted a clear differentiation between the two species, regardless of the isolates' country of origin. The modified MIDI method gave the most consistent and reproducible fatty acid results. The failure of the MIDI method to differentiate between R. oryzae and R. oryzae-sativae isolates suggests that the 30 minutes saponification step is insufficient to completely break the cell wall of these two species. This study demonstrated that fatty acid profiles, obtained by the modified MIDI protocol, have the potential as a diagnostic tool for both R. oryzae and R. oryzae-sativae.