ABSTRACT: Purpose:Aspergillosis is an important fungal disease affecting millions of individuals worldwide. The genus of Aspergillus consist of various complexes, causing a wide spectrum of diseases from superficial infections in immunocompetent hosts to life-threatening disseminated infections among immunocompromised patients. This study aimed to identify Aspergillus species by phenotypic (total isolates) and molecular tests (35 isolates), obtained from patients in Isfahan (the third-largest city of Iran) between 2010 and 2018, and determine the susceptibility of 35 clinical isolates to itraconazole (ITR), amphotericin-B (AMB), and voriconazole (VOR). Patients and Methods:Based on clinical signs, a total of 2385 suspected cases were included in this retrospective study from January 2010 to December 2018. Direct microscopic examination with potassium hydroxide, sabouraud dextrose agar with chloramphenicol, and czapekdox agar media was applied to identify etiologic agents. Thirty-five Aspergillus species collected from January 2016 to December 2018 were identified by PCR-sequencing of ITS1-5.8SrDNA-ITS2 region, and their susceptibility to ITR, AMB, and VOR was determined using E-test. Results:Based on direct microscopy and positive culture, 132 out of 2385 suspected cases had Aspergillus infection (5.5%). Fifty-four patients were male, and 78 patients were female. Patients in the age groups of 41-50 and 21-30 years had the highest and lowest frequencies, respectively. Aspergillus flavus/oryzae (n=54), A. fumigatus (n=24), A. niger (n=15), and A. terreus (n=12) were the most prevalent Aspergillus species, respectively. Among 35 Aspergillus species, the MIC ranges of AMB, ITR, and VOR for A. flavus/oryzae, A. niger, and A. terreus were (0.5-4 ?g/mL; 0.5-16 ?g/mL; 0.25-8 ?g/mL), (1 ?g/mL, 1 ?g/mL, 1 ?g/mL), and (4-4 ?g/mL, 0.5-1 ?g/mL, 0.5-1 ?g/mL), respectively. Conclusion:Aspergillus infections have a wide spectrum of clinical manifestations and often occur in immunocompromised patients. Accurate identification at the species level is essential since the emergence of cryptic species is connected to different patterns of AFST that affect patient treatment outcomes. Azole-resistant Aspergillus spp. is a global concern, and the detection of the route of resistance is pivotal to prevent and control infection.