The Aspergillus species are main causative agents of various infections such as invasive aspergillosis (IA) in immunocompromised patients and these infections have high mortality rates. In this study, we provide insight in the species causing aspergillosis in Iran based on morphology and sequence data. Clinical ( n  = 117) and environmental isolates ( n  = 54) collected during 2010–2016 from University hospitals in Mashhad and Tehran (Iran) were identified both morphologically and molecularly using partial calmodulin ( CaM ) gene sequences. Clinical cases were identified based on EORTC/MSG criteria. Aspergillus flavus ( n  = 96, 55%) was the most prevalent species among the clinical and environmental isolates while A. fumigatus ( n  = 13, 7.5%) ranked fourth after A. tubingensis ( n  = 23, 13%) and A. welwitchiae ( n  = 18, 10%). Species such as A. tubingensis , A. welwitschiae , A. fumigatus , A. sydowii , A. neoniger and A. terreus were present in both clinical and environmental samples indicating the possible environmental source of infections. Interestingly, A. niger was isolated only once. Furthermore, 13 other rare and cryptic Aspergillus species were detected. Pulmonary and respiratory disorders ( n  = 33), followed by transplantation ( n  = 23), invasive fungal rhinosinusitis ( n  = 14), and haematological malignancies ( n  = 12) were major predisposing factors. According to EORTC/MSG criteria, there were 43 probable cases identified followed by 36 cases for each of proven and possible ones. Correct molecular identification will be useful for further epidemiological studies.