45S ribosomal DNA loci are organized as long head-to-tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure, the rDNA loci are the major sources of gaps in genome assemblies, and expression status of particular arrays remains elusive. We combined cytogenomics techniques with bioinformatics to reconstruct individual 45S rDNA arrays in hexaploid wheat, thus building a stepping stone for locus-specific analyses. The engagement of particular loci in rRNA transcription was assessed across several tissues and the locus-specific transcription was compared with cytosine methylation level in corresponding rDNA units. Results of the analyses allowed us to outline a scenario of the evolutionary histories of rDNA loci in three wheat subgenomes.