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Purpose: Otomycosis is a mycotic infection of the external auditory canal and can be caused by a wide range of fungal species. In this study, we aimed to identify fungal isolates from patients suspected of otomycosis., Methodology: External ear canal samples were taken from patients referred to the outpatient department of Shahid-Mofatteh Clinic in the city of Yasuj, Iran, and examined by direct microscopy and culture. DNA of the isolated fungi was tested by internal transcribed spacer PCR restriction fragment length polymorphism analysis for identification of yeasts and β-tubulin sequencing for identification of Aspergillus species., Results: Among 275 patients suspected of otomycosis, 144 cases (83 female and 61 male) were confirmed with otomycosis. For 89% (n=128) of positive cultures, microscopy was also positive, while there were no cases with a microscopy-positive and culture-negative result. The predominant predisposing factor was self-cleaning of the external ear using unhygienic tools, and the main risk occupation was 'housewife'. The most common isolated fungi were typically Aspergillus (n=120), including 73 isolates of Aspergillus section Nigri, 43 of section Flavi, 3 of section Terrei and 1 of section Fumigati. After sequencing, 44 out of 73 strains primarily identified as Aspergillus niger turned out to be Aspergillus tubingensis. Thirty-five isolates were identified as Candida, including Candida parapsilosis (n=22), Candida albicans (n=12) and Candida tropicalis (n=1)., Conclusion: Aspergillus tubingensis was the most common species involved in otomycosis. This work corroborates the difficulty of precise identification of species within the black Aspergilli by morphological characteristics.