Abstract Aspergillosis is one of the most common human fungal infections. The invasive form of this infectious disease has high mortality rates. Moreover, antifungal resistance has been increasing, thereby limiting treatment options. In Iran, limited species distribution, genotyping and susceptibility data regarding aspergillosis is available, especially during the COVID-19 pandemic. In the current study, 124 patients with proven (n = 31), probable (n = 24) and possible (n = 46) aspergillosis and aspergillus colonization (n = 19) were investigated. Isolates were identified to species level based on calmodulin sequencing. Antifungal susceptibility testing was performed with microbroth dilution against common antifungal agents, i.e. amphotericin B, azoles and echinocandins. Additionally, short tandem repeat genotyping was conducted on common Aspergillus species to assess genetic relatedness. Aspergillus flavus was the most common species for proven aspergilossis cases, followed by identification of single cases of A. fumigatus, A. terreus, A. niger. These Aspergillus species were also mostly found in other patients with probably aspergillosis, in addition to four cases of possible aspergillosis with rare or cryptic species A. candidus, A. citrinoterreus, A. tubingensis and A. fumigatiaffinis, respectively. Using available epidemiological cutoff values (ECVs) no isolates were non-wild type to the tested antifungal drugs, while the A. fumigatiaffinis and A. citrinoterreus isolate demonstrated reduced susceptibility to respectively amphotericin B and Itraconazole, and amphotericin B only. With high-resolution short tandem repeat genotyping, several A. flavus clusters were found and their spatial and temporal clustering suggested nosocomial origins. To conclude, aspergillosis cases in Iran were caused by diverse but susceptible species, with A. flavus being dominant and associated with several events of potential nosocomial transmission.