Viola caspia subsp. sylvestrioides, a small important medicinal herb, belongs to the subsection Rostratae (sec. Viola) of genus Viola L. This study was performed to develop an efficient protocol for in vitro regeneration of V. caspia subsp. sylvestrioides as an ex situ technique for conservation of endangered plants and extraction of secondary metabolites. Leaf and petiole explants were cultured on half and full-strength Murashige and Skoog (MS) media supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), isopentenyl adenine (2ip), thidiazuron (TDZ), and naphthalene acetic acid (NAA). Direct organogenesis was induced on half and full-strength MS media supplemented with TDZ (0.7 to 3.5 mg/l) from leaf and petiole explants. Indirect shoot organogenesis was induced on half-strength MS supplemented with 3.5 mg/l 2ip and 2.5 mg/l NAA followed by transferring the obtained callus onto a half-strength MS containing 0.5 mg/l BAP. The highest frequency of shoot organogenesis (100 and 86.66%) was noted for petiole and leaf explants, respectively, on halfstrength MS medium supplemented with 2.8 mg/l TDZ (approx. 7.66 and 4.33 shoots per petiole and leaf explant, respectively). The induced shoots were used for root induction on a half-strength MS medium with 0.5 mg/l indole- 3-butyric acid (IBA). Phytochemical constituents in leaf tincture were determined by gas chromatography/ mass spectrometry (GC/MS) analysis. GC/MS results revealed that vitamin E was the main component in the tincture of Viola leaves. The present study provides a simple and rapid protocol for in vitro regeneration of V. caspia subsp. sylvestrioides plants which can be used for gene transfer and conservation purposes, and in pharmaceutical studies.