T. T. Tzatzani, I. Michail, I. Samartza, N. Krigas and G. TsoktouridisSt. John's wort (Hypericum perforatum) is a perennial species with a worldwide interest due to its pharmaceutical characteristics which is also of ornamental value for gardening with native elements. Given that in vitro culture allows mass multiplication of selected genotypes, we investigated the micropropagation of the ex situ cultivated Greek native H. perforatum subsp. veronense using novel substances and its genetic fingerprinting. Apart from common auxins and cytokinins, a culture medium (BB) was used for the first time for its micropropagation and the effect of betaine, PVP, SNP and triacontanol were explored for the first time. Betaine addition of 200 mg/L produced proliferated shoots and roots without the need of plant growth regulators although a 0.5 mg L-1 BA was the best treatment in proliferation experiments. Shoot number was highest in 0.5 BA + 0.1 IBA (mg L-1), in 1 μΜ sodium nitroprusside (SNP) + 1 mg L-1 IAA+200 mg L-1 betaine and in PVP (0.5-1.5 mg L-1) + 1 mg L-1 IBA + 200 mg L-1. Greater SNP concentration had either no differences or negative effect, while the greatest root number were recorded in Control. All three treatments of triacontanol (1-10 μg L-1) + 0.5 mg L-1 BA + 200 mg L-1 betaine resulted in higher shoot number. However, the statistically greatest root number were recorded in low concentration of SNP, or in absence of PVP and triacontanol. The micro-plants of H. perforatum subsp. veronense obtained from the SunCapTM sealed vessels and transplanted into the soil substrates presented 90.2% success; they were then transplanted to 1 L pots and were successfully established in the field for cultivation. The molecular characterization of H. perforatum subsp. veronense was achieved with DNA barcoding fingerprints deposited in the GenBank regarding four molecular markers of cpDNA (petB/petD, rbcL, trnL/trnF and rpoC1).