We truncated the short arm of chromosome 3 to delete the aflatoxin biosynthesis gene homolog cluster using telomeric repeats in Aspergillus oryzae. The predicted deletion was confirmed by Southern blot analyses. This telomere-mediated chromosomal truncation method enables the development of an artificial chromosome in A. oryzae. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.