Rhizopus oryzae is the most frequent causative agent of zygomycosis. Although zygomycosis causes considerable morbidity and mortality in immunocompromised patients, the epidemiology of the disease is not well studied and no standard molecular typing method has been described for any of the causative agents. Here we describe a multilocus microsatellite typing (MLMT) method for R. oryzae. R. oryzae genome sequences were downloaded from the Fungal Genome Initiative database (Broad Institute). The intergenic regions and ORFs of approximately 5.7 Mb were screened for repeat regions with the help of the online repeat search tool Repeat Masker. Of the 30 microsatellite loci identified, 3 microsatellites [RO3, (CCT)(n); RO4, (TA)(n); and RO8, (GAA)(GGA)(n)] were selected after validation of the ability to amplify them and their size variation in 8 randomly selected clinical isolates of R. oryzae. Nucleotide sequence analysis of these loci demonstrated polymorphism in the microsatellite repeat number. The capabilities of these microsatellite loci were assessed for strain differentiation on 30 clinical isolates, based on fragment size determination in an automated capillary electrophoresis using fluorescent labelled primers. These three polymorphic microsatellite loci were found to have good discriminatory power (D) (RO3, D=0.846; RO4, D=0.747; RO8, D=0.742; with a combined D=0.986) and stability for seven subcultures. It was also confirmed that the MLMT method may be applied to both R. oryzae and Rhizopus delemar (a proposed new species), although MLMT analysis could not differentiate them into two clusters. The MLMT system, described here for what is believed to be the first time for a zygomycotic fungus, holds promise as a powerful tool for the strain typing of R. oryzae.