Background Microsatellites (or short tandem repeats, STRs) are the genetic markers of choice for studying Aspergillus fumigatus molecular epidemiology due to its reproducibility and high discrimination power. However, the specificity of these markers must be investigated in a group of isolates from closely related species. The aim of this work was to test a microsatellite-based PCR multiplex previously designed for A. fumigatus in a set of species belonging to section Fumigati, namely Aspergillus fumigatiaffinis, Aspergillus lentulus, Aspergillus novofumigatus, Aspergillus unilateralis, Aspergillus viridinutans, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae. Results The reference A. fumigatus strain ATCC 46645 was easily genotyped in standard conditions showing a final electrophoretic profile of 8 expected peaks corresponding to each microsatellite locus. Inversely, no peaks were observed for all other species from section Fumigati, with an exception for marker MC6b in A. unilateralis. By screening the genome sequence of Neosartorya fischeri NRRL 181, the results showed that MC3, MC6a and MC7 might be employed for N. fischeri genotyping since these markers present several repeats of each motif. The accumulation of insertions and deletions was frequently observed in the genomic regions surrounding the microsatellites, including those where the A. fumigatus primers are located. The amplification of microsatellite markers in less stringent amplification conditions resulted in a distinct electrophoretic profile for species within section Fumigati. Conclusions Therefore, the microsatellite-based PCR multiplex allow simple identification of A. fumigatus and, with a slight modification of temperature conditions, it also allows discriminating other pathogenic species within section Fumigati, particularly A. fumigatiaffinis, N. fischeri and N. udagawae.