Employing FISH analysis as well as BLAST and CUSTAL W (1.82) programs, we investigated types of DNA nucleotide sequences building an additional heterochromatic band in 2R chromosomes of 3 lines of Secale vavilovii Grossh. The probes used in FISH analysis were designed based on the reverse transcriptase sequence of Ty1-copia and Ty3-gypsy retrotransposons and the 5S rRNA gene sequence. No hybridization signals from the reverse transcriptase probes were observed in the chromosome region where the additional band occurs. On the other hand, signals were observed after hybridization with the 5S rDNA probe, clearly suggesting the presence of that type of sequences in the analyzed heterochromatin band. Using BLAST and CUSTAL W programs, we revealed high similarity of the JNK1 sequence to the 5S rRNA gene from Hordeum chilense (HCH1016, HCH1018, 88%) and to a fragment of the 5S rRNA sequence of H. marinum (HMAR003, 97%). In addition, the same fragment of JNK1 was shown to be very similar to the part of the Angela retrotransposon (92%) as well as to the SNAC 426K20-1 transposon (89%) belonging to CACTA family, both from Triticum monococcum, and to Zingeria biebersteiniana pericentromeric sequences (78%). The similarity of JNK1 to those sequences may be accidental or the JNK1 may represent an ancient mobile genetic element that caught the 5S rRNA sequence. During the evolution those sequences might have been accumulated in the particular region on the 2R chromosome. Our results suggest that the additional heterochromatin band in chromosomes 2R of S. vavilovii is a collection of defective genes and/or mobile genetic elements.