Abstract: Using sequence-tagged DNA sequencing on the Roche 454 platform, we studied intragenomic polymorphism of one of the 35S rRNA regions (18S rDNA fragment–ITS1–fragment of 5.8S rDNA) in three hexaploid Avena species with karyotypes AACCDD and a tetraploid species A. insularis (AAСС or CCDD). Instead of expected 50% of C-variant ITS1 in A. insularis and 33% of C-variant ITS1 in hexaploids A. fatua, A. ludoviciana, and A. sterilis, the actual rate of C-subgenome specific ITSs comprised around 3.3% of rDNA in A. insularis and 1.4–2.4% of rDNA in hexaploid genomes. The 18S rDNA (fragment), ITS1 and 5.8S rDNA (small fragment) of the C-subgenome origin were 10 times more variable than the same sequences from the A-genome. Some of the C-subgenome sequences contained deletions, including deletions in the 18S rRNA coding region. The results of FISH hybridization with pTa71 and pTa794 confirm the fact that polyploids lost a significant part of the 35S rDNA and 5S rDNA obtained from a diploid ancestor with the CC karyotype. Our results show that the loss of the 35S rDNA of the C type occurs against the background of multiple single nucleotide polymorphisms (SNPs) and deletions accumulation in these sequences. The fact that all C-subgenome ITS1 sequences in the genomes of polyploids were represented by single (unique) copies might indicate that the appearance of multiple mutations in the “repressed” 35S rRNA loci was not accompanied by homogenization of rDNA. Hence, there is a reason to believe that the process of rDNA isogenization and the process of transcription/silencing are related phenomena.