Objectives Rhizopus arrhizus is recognized as an emergent agent of superficial and invasive mucormycosis. Despite an increasing number of these infections, the molecular epidemiology of Rhizopus species has not been well studied. Materials and methods In this study, 43 R. arrhizus strains (25 environmental and 18 clinical isolates) were genotyped using six novel panels of microsatellite markers. Results Upon the analysis of 43 isolates, 4-8 distinct alleles were detected for each marker. The discriminatory power for the individual markers ranged from 0 center dot 522 to 0 center dot 830. The combination of all six markers yielded 33 different haplotypes with a high degree of discrimination (0 center dot 989 D value). A four-marker combination were selected as the most parsimonious panel achieving D > 0 center dot 95. One clinical isolate and one environmental isolate shared the same genotype suggesting the possible nosocomial outbreak of mucormycosis in hospitalized patients. We have noted that the strains isolated from cutaneous mucormycosis were different from the strains isolated from rhino-orbito-cerebral mucormycosis. Then, the hypothesis of particular tropism of infectious strains for a given site is not excluded. The standardized indices of association IA and rBarD were significantly different from zero (P < 0 center dot 01), suggesting a prevailing clonal reproduction. The environmental population was significantly differentiated from clinical populations (Fst = 0 center dot 2249). Conclusions Microsatellite typing method described in our study showed an excellent degree of discriminatory power. It is a promising tool for illuminating the molecular epidemiology of R. arrhizus species, including strain relatedness and transmission pathways.