Thirteen Aspergillus isolates recovered from nails of 13 patients (fingernails, n = 2; toenails, n = 11) with onychomycosis were characterized. Twelve strains were identified by multilocus sequencing as Aspergillus spp. ( Aspergillus sydowii [n = 4], Aspergillus welwitschiae [n = 3], Aspergillus terreus [n = 2], Aspergillus flavus [n = 1], Aspergillus tubingensis [n = 1], and Aspergillus unguis [n = 1]). Isolates of A. terreus , A. flavus , and A. unguis were also identifiable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The 13th isolate (HKU49 T ) possessed unique morphological characteristics different from other Aspergillus spp. Molecular characterization also unambiguously showed that HKU49 T was distinct from other Aspergillus spp. We propose the novel species Aspergillus hongkongensis to describe this previously unknown fungus. Antifungal susceptibility testing showed most Aspergillus isolates had low MICs against itraconazole and voriconazole, but all Aspergillus isolates had high MICs against fluconazole. A diverse spectrum of Aspergillus species is associated with onychomycosis. Itraconazole and voriconazole are probably better drug options for Aspergillus onychomycosis.