AbstractThree out of four RNA components of ribosomes are encoded by 45S rDNA loci, whose transcripts are processed into 18S, 5.8S and 26S ribosomal RNAs. The loci are organized as long head-to-tail tandem arrays of nearly identical units spanning over several megabases of sequence. Due to this peculiar structure, the exact copy number, sequence composition and expression status of the 45S rDNA remain elusive even in the genomics era, especially in complex polyploid genomes harbouring multiple loci. We employed a novel approach to study rDNA loci in polyploid wheat, comprising chromosome flow sorting, optical mapping and high-throughput rRNA sequencing. This enabled unbiased quantification of rDNA units in particular arrays. Total number of tandemly organised units in wheat genome was 4390, with 64.1, 31.4, 3.9 and 0.7% located in short arms of wheat chromosomes 6B, 1B, 5D and 1A, respectively. Optical maps revealed high regularity of tandem repeats in 1B and 5D, while the 6B array showed signatures of non-rDNA sequences invasions; 1A locus harbored highly rearranged repeats with many irregularities. At the expression level, only 1B and 6B loci contributed to transcription at roughly 2:1 ratio. The 1B:6B ratio varied among five analysed tissues (embryo, coleoptile, root tip, primary leaf, adult leaf), being the highest (2.64:1) in adult leaf and lowest (1.72:1) in coleoptile. In conclusion, a fine genomic organisation and tissue-specific expression of wheat rDNA loci was deciphered, for the first time, in a complex polyploid species. The results are discussed in the context of rDNA evolution and expression.