This study addresses the usefulness of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for reliable identification of the two most frequently occuring clinical species of Rhizopus, namely R. arrhizus with its two varieties arrhizus and delemar and R. microsporus. The test-set comprised 38 isolates of clinical and environmental origin previously identified by ITS sequencing of rDNA. Multi-locus sequence data targeting three gene markers (ITS, ACT, TEF) showed two monophylic clades for R. arrhizus and R. microsporus (bootstrap values of 99%). Cluster analysis confirmed the presence of two distinct clades within R. arrhizus representing its varieties arrhizus and delemar. The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the distinction between R. arrhizus and R. microsporus and the presence of two varieties within the R. arrhizus. An in-house database of thirty reference main spectra (MSPs) was initially tested for correctness using commercially available databases of Bruker Daltonics. By challenging the database with the same strains of which an in-house database was created, automatic identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety level. Based on the Principal Component Analysis (PCA), two MSP dendrograms were created and showed concordant with the multi-locus tree thus MALDI-TOF MS is a useful tool for diagnostics of mucoralean species.