Wide hybridization expands the gene pool of cultivated species and can produce genotypes with practical value, often resistant to diseases. In plants, ISSRs are often used for hybrid identification as well as for diversity evaluation. We estimated genetic similarity among advanced genetic stocks of triticale hybrids and identified Aegilops juvenalis introgressions into triticale. Fourteen ISSR primers amplified from 12 to 23 DNA fragments. The number of polymorphic products per primer ranged from three to nine, with a mean of 5.1. The polymorphic information content values ranged from 0.39 to 0.66, with a mean of 0.52. Thirteen primers amplified products indicating presence of A. juvenalis chromatin. The largest mean genetic similarity (0.84) with all other forms was shown by a strain derived from an A. juvenalis 6x × [(Lanca × L506/79) × CZR142/79] hybrid, while A. juvenalis was the least similar (0.33). We conclude that ISSRs can reliably identify A. juvenalis chromatin in the triticale background and efficiently estimate genetic similarity of hybrids and parental forms.