Objective To study the distribution of fungal species in otomycosis, and to explore the method of multiplex polymerase chain reaction (PCR) to detect and identify common fungal species in otomycosis.Methods Specimens were collected from external ear canal of 137 patients with otomycosis who visited the department of otolaryngology, Jingzhou central hospital from March 2022 to June 2023. Part of the specimen was routinely cultured with modified Martin solid medium. After culture, DNA was extracted, and the fungal species were identified by PCR amplification and sequencing. At the same time, the other part of the same specimen was directly extracted by DNA, and multiple PCR were performed twice, using the universal and specific fungal primers respectively. The fungal species could be identified according to the length of bands obtained by agarose gel electrophoresis.Results A total of 128 Aspergillus strains (93.4%) were identified by traditional methods and multiplex PCR in 137 otomycosis specimens, including 73 strains of Aspergillus terreus, 24 strains of Aspergillus flavus, 18 strains of Aspergillus niger, and 13 strains of Aspergillus fumigatus. There were 9 strains (6.6%) of Candida albicans. The traditional method took 4 to 7 days, while the multiplex PCR technique could identify the fungal species in 6 to 8 hours. The results of the two methods were compared, and the coincidence rate was 100%.Conclusions The most common fungal species in patients with otomycosis in this region are Aspergillus and Candida. In addition to common species, multiplex PCR can also be used to identify rare species such as Candida auris and Candida parapsilosis with high drug resistance and high mortality. Compared with traditional methods, multiplex PCR has the advantages of rapid and accurate detection and identification of otomycosis, which is worthy of clinical promotion.