Physalis ixocarpa commonly called tomatillo belongs to the genus Physalis and family solaneaceae. There are about seventeen accession of P. ixocarpa reported from the world. We are reporting for the first time new accession of P. ixocarpa from district Shangla KPK Pakistan, with molecular biochemical and in vitro culture study for their pharmacological importance and conservation. The introduction of new accessions in the genus Physalis was supported by morphometric and molecular data of sixteen accessions provided by National plant germplasm system USDA (USA). Seventeen accessions were evaluated for nine morphometric and two physiological traits. Among these traits, plant height, relative water content, total chlorophyll content and leaf area were found as best for the differentiation among these accessions. The data regarding these traits were analyzed by NTSYS software using UPGMA method. The morphometric similarity coefficient between different accessions was 0.15 to 0.61. These accessions were also screened for genetic differentiation using RAPDs primers. Out of 32 primers, only 8 primers amplified the DNA fragments, and provided genetic information about these accessions. The genetic similarity among different accessions was in the range of 0.03 to 0.33. Genetic similarity matrix showed maximum genetic similarity of 33% between two combinations (PI662845 and PI662846, PI512909 and PI66843) and minimum genetic similarity of 3% obtained between PI512005 and PI360740. The RAPD data supported the ecological distribution and relation among the isolated geographic population. The topology of dendogram indicated that most of the accessions with similar geographic condition were grouped together in dendogram. Mexican and American accessions were clustered together in separate groups. The new accession of P. ixocarpa from Pakistan was clustered together with one accession of Indian and American origin in the dendogram. It is inferred from molecular typing that accessions PI512005 and PI360740 were the most diverse accessions among the different accessions of P.ixocarpa. The medicinal importance and economic value was investigated by preliminary bioactivities and the recovery of bioactive compounds of the accession. The variation in bioactivities of the different parts of the plant is due to uneven localization and distribution of secondary metabolites in plant matrix. Among different parts of the plant tested for antimicrobial activity, calyx showed inhibitory activity against most of bacteria. Antifungal activity was found only in leaf part of the plant. Strong antioxidant activities were found in different parts of the plant. Crude methanolic extract from leaf, stem, fruit, and water fraction from stem exhibited strong antioxidant activity as compared to other samples. Phyto-chemical screening showed the presence of active compounds (alkaloid, polyphenol, tannin, glycoside) responsible for the bioactivities of the plant parts. Polyphenol of dietary and pharmaceutical interest was quantified in different parts of the plant through HPLC and spectrophotometric 2 assay. Total high polyphenol content was found in leaf followed by stem, calyx and fruit. High concentration of gallic acid was observed in the leaf followed by fruit, stem and calyx. The newly reported accession of P. ixocarpa has now become endangered due to overgrazing, erosion, over exploitation and limited availability. The micro propagation protocol was optimized in the present study. The data regarding the in vitro culture of the subject plant indicated that maximum number of shoots was obtained on full strength shooting media containing 1.5 mg.L-1 BAP and maximum number of primary and secondary roots were produced on half strength media supplemented with 1mg.L-1IBA for micro propagation to maintain the biodiversity of this endangered medicinal plant.