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ABSTRACTAspergillussection Nigriincludes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two black Aspergillusstrains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and their in vitrotriazole susceptibilities investigated. Aspergillus tubingensiswas isolated from humans, animals, and environmental settings, whereas Aspergillus awamoriand Aspergillus nigerwere isolated exclusively from humans. Phylogenetic analyses of ß-tubulin and calmodulin gene sequences were concordant in differentiating A. tubingensisfrom A. awamoriand A. niger. Voriconazole and posaconazole (PSZ) were the most active triazoles. One A. tubingensisstrain was resistant to itraconazole and PSZ and one A. nigerstrain to PSZ. Sequence analysis of the cyp51Agene revealed different sequence types within a species, and A. tubingensisstrains were also phylogenetically distinct from A. awamori/A. nigerstrains according to the strain origin and susceptibility profile. Genetic analysis of the cyp51Asequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in black Aspergillusisolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. The cyp51Asequence is useful for the molecular identification of black Aspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena.