Aims Aspergillus sp. are ubiquitous saprophytic fungi and their conidia easily inhaled. This is particularly important in immunocompromised patients, more susceptible to developing invasive aspergillosis. In addition to A. fumigatus sensu stricto, cryptic species, many resistant to antifungal drugs, have been associated with invasive infections, making it important to assess their presence and diversity in different environments. Therefore, the aim of this study was to assess the presence, diversity and susceptibility to antifungal drugs of airborne fungi. Moreover, in azole-resistant A. fumigatus sensu stricto isolates the presence of underlying molecular mechanisms of resistance was investigated. Methods and Results Eighty-four Aspergillus isolates were collected from the environment air in hospitals and the Water Treatment Plant. The use of molecular tools allowed to detect 12 different cryptic species, showing a prevalence of 21 center dot 4%. The majority of isolates (69 center dot 0%) belonged to A. fumigatus complex and 65 center dot 4% were A. fumigatus sensu stricto. Among these, 21 center dot 8% were resistant to itraconazole (ITZ), 38 center dot 2% to posaconazole and 87 center dot 3% to isavuconazole; none of them were resistant to voriconazole or amphotericin B. Sequencing of the cyp51A gene on the 12 A. fumigatus sensu stricto ITZ-resistant isolates revealed the presence of mutations. Conclusion Our study reports a large number of environmental-resistant Aspergillus species, including A. fumigatus sensu stricto that display an important role in invasive fungal infections. None of the environmental isolates showed mutations on cyp51A gene related to azole resistance. Significant and Impact of the Study This study is the first assessment of molecular resistance mechanisms in A. fumigatus sensu stricto environmental isolates, in Portugal. Since TR34/L98H and TR46/Y121F/T289A cyp51A mutations were already reported in the clinical setting in Portugal (Monteiro et al. J Glob Antimicron Resist 13: 190-191, 2018; Pinto et al. Front Microbiol 9: 1656, 2018), and have been linked to environmental route, it is utmost importance to perform surveillance network for azole-resistant A. fumigatus.