The recent increase in azole-resistant Aspergillus fumigatus is a global concern. Identifying the mutations that confer azole resistance is essential for developing novel methods for prompt diagnosis and effective drug treatment. We screened A. fumigatus clinical isolates for novel mutations conferring azole resistance. We compared the genomic sequences of susceptible and resistant isolates without mutations in cyp51A (non-cyp51A) and found mutations in hmg1 and erg6 involved in ergosterol biosynthesis. We also found the novel mutations in these genes in azole-resistant isolates with different genetic backgrounds. The resistant isolates with mutations in hmg1 showed increased intracellular ergosterol levels compared with susceptible isolates. This finding supports the concept that the ergosterol level is a determinant for resistance to any class of azoles. Multiple isolates with increased resistance to azole possessed a mutation in hmg1, indicating that this mutation is widely present in non-cyp51A azole-resistant A. fumigatus.