The Crucifers family (Brassicaceae) includes a large number of economically important crops, particularly Brassica rapa , which is a widely used model plant for molecular genetic studies of oilseeds. B. rapa is a highly polymorphic species that includes many of genetically distinct subspecies. Considering this fact, the intraspecific hybridization of B. rapa subspecies is considered a promising breeding approach aimed at increasing the genetic diversity of the crop. Previously, the authors have shown that one of such hybrids, B. rapa subsp. oleifera f. biennis × (subsp. rapifera × pekinensis ), could be a valuable oil feedstock due to its increased productivity. However, obtaining hybrids and their subsequent breeding would require the involvement of various molecular marker systems. So far, the method of estimating the length polymorphism of the first (TBP) and second (cTBP) introns of β-tubulin has demonstrated its high accuracy and reliability in the identification (DNA-barcoding) of flowering plant taxonomic units at different levels. In the present study, the productivity of such hybrid oil tyfon ( B. rapa subsp. oleifera f. biennis × (subsp. rapifera × pekinensis )) was evaluated and DNA-barcoding of different hybrid tyfon lines ( B. rapa subsp. oleifera f. biennis × (subsp. rapifera × pekinensis )) and its parental B. rapa subspecies using the β-tubulin intron length polymorphism assessment approach was carried out. Based on the data of the molecular genetic analysis, which included the assessment of length polymorphism of the first and second introns of β-tubulin genes, we were able to confirm the origin of the oil tyfon hybrid ( B. rapa subsp. oleifera f. biennis × (subsp. rapifera × pekinensis )) hybrid from Dutch leaf tyfon ( B. rapa subsp. rapifera × pekinensis ) and winter turnip ( B. rapa subsp. oleifera ) with high confidence. Along with that, it was possible to differentiate var. glabra and var. laxa accession of napa cabbage ( B. rapa subsp. pekinensis ) for the first time using combined TBP and cTBP analyses. A variation in the number of amplified regions of β-tubulin introns was noted in different genotypes; however, these differences did not appear to be a specific feature of a particular subspecies/hybrid. This suggests that B. rapa hybrids most likely do not differ in ploidy compared to their parental genotypes. In addition, it was shown that the mentioned oil tyfon hybrid lines of Ukrainian breeding show a significant level of morphological variation despite their common breeding pedigree.