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We present findings on the nucleosomal arrangement in the genome of the basidiomycete Mixia osmundae, focusing on nucleosomal linker DNA regions. We have assembled the genomic sequences of M. osmundae, annotated genes and transcription start sites (TSSs) on the genome, and created a detailed nucleosome map based on sequencing mono- and dinucleosomal DNA fragments. The nucleosomal DNA length distribution of M. osmundae is similar to that of the filamentous ascomycete Aspergillus fumigatus, but differs from that of ascomycetous yeasts, strongly suggesting that nucleosome positioning has evolved primarily through neutral drift in fungal species. We found clear association between dinucleotide frequencies and linker DNA regions mapped as the midpoints of dinucleosomes. We also describe a unique pattern found in the nucleosome-depleted region upstream of the TSS observed in the dinucleosome map and the precursor status of dinucleosomes prior to the digestion into mononucleosomes by comparing the mono- and dinucleosome maps. We demonstrate that observation of dinucleosomes as well as of mononucleosomes is valuable in investigating nucleosomal organization of the genome.