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Peach is a popular sweet fruit with a very good climate adaptation and high production. It offers many health benefits determined by its biochemical composition. However, sensitive people can be sensitised by Pru p 3 non-specific lipid transfer protein family directly or through cross reaction as a member of Bet v 1 homologues. The majority of research is focused on the protein, less data can be found in genomics or transcriptomics. This study performed RFLP analysis by chosen restricted enzymes (BfaI, MseI, NlaIII) for non-coding region of Pru p 3 (NCBI: KC311811.1) as a tool to distinguish closely related isoforms of the allergen. BfaI cut amplicon into 5 fragments corresponding to the Yulu variety in silico cleavage and polymorphism was not detected. For MseI and NlaIII polymorphisms were found in the cleavage sites, two types of restriction profiles were created for both. None of the NlaIII profiles corresponds to the restriction profile of in silico cleavage. The study confirms the varietal differences in Pru p 3 gene and supports a hypothesis that allergenicity depends on both qualitative and quantitative factors that are different and specific to each variety.