We identified 5 975 567 mono- and 6 995 122 di-nucleosome positions of the fungus Aspergillus fumigatus, which was detected at high resolution based on the DNA sequence data obtained from both mono- and di-nucleosomal DNA fragments. We show that the distribution of lengths of the mono-nucleosomal DNA fragments has two peaks at 135 and 150 nt, whereas the distribution of di-nucleosomal DNA fragment lengths has a single peak at 285 nt. Although the gene bodies of the active and inactive genes and the inactive gene promoters had the two peaks of the mono-nucleosomal DNA fragment lengths, the active gene promoter lost the longer peak at 150 nt. Our findings strongly suggest that the nucleosomes protecting longer DNA fragments against MNase at the promoters, thereby inhibiting high gene expression.