Tissue cultured Pimpinella tirupatiensis plantlets were subjected to assessment of genetic stability considering the fact that associated in vitro stress might result in breakdown of control mechanism causing instability of the genome. We have used two DNA based molecular markers to assess the genetic fidelity of in vitro regenerated Pimpinella tirupatiensis through shoot tip from in vitro raised seedlings. The shoot tips upon transfer to MS medium containing different concentrations and combinations of 6- benzyl aminopurine (BAP) kinetin (KN), 2- isopentyl adenine (2- ip), α- napthalene acetic acid (NAA) and indole 3- acetic acid (IAA). The best morphogenic response was observed on MS medium fortified with BA (13.31 µM) and NAA (2.69 µM) which exhibited the highest regeneration frequency (90%), the maximum number of shoots/explants (6.50 ± 0.91) and shoot length (3.20 ± 0.20) within 5 weeks. Rooting was achieved within 15 days of shoot implantation on ½ strength MS media fortified with BAP (13.31 µM) and IBA (9.8 µM). The rooted plantlets were successfully acclimatized with 85% survival rate. Out of 20 RAPD and 3 ISSR primers screened, only 6 random amplified polymorphic DNA (RAPD) and all three inter simple sequence repeats (ISSR) primers produced clear reproducible and scorable bands. All banding profiles from micropropagated plants were monomorphic and similar to the mother plant indicating an absence of noticeable genetic variation in the regenerated plantlets. This study is of high significance as these could be commercially utilized for large scale production of true-to-type plantlets in Pimpinella tirupatiensis.