The Biodiversity assessment using ethnobotanical and ecological inspection complemented by genetic variation investigation of tree Juniperus excela was undertaken in Zarghoon Juniper Forest during the years 2009-11. In contemporary study the illustrious data assortment procedure entitled Participatory Rural Appraisal (PRA)-Type Participatory Assessment of Natural Resources (PANR) was applied. PRA tools social map, transit walk, structured, semi structured interviews and pie diagrams were used. PRA was piloted in five villages as central habitat of five constellations embraced of total 17 villages in Zarghoon Juniper ecosystem.Meetings with local communities both male and female separately were conducted and PRA tool carried out using charts and markers. Ethnobotanical assessment revealed that 60%, 35% and 5% medicines were used orally, topically and boiled to inhale, respectively. Correspondingly, in case of parts of plants usage 57, 26, 10 and 7% leaves, seeds, flower and roots were used, respectively. The socio-economic condition of the area indicated local community was deficient of basic amenities of life. The dependency on natural resources was high and unplanned land fragmentation occurred as the major source of income (90%) was from agriculture and livestock rearing and only 10% income by trade, services and labor. Rate of deforestation assessed by counting cut stumps, ranged from 6.69 to 4.70/acre. Average Diameter at Breast Height (DBH) in inches above ground at 4.5 measured and that ranged from highest 37.13 to lowest as 29.40. Goat made the dominant livestock population of 75%, sheep 23% and donkey 2%. Sex ratio of J. excelsa showed predominance of male with a mean value of 103±23ha-1 (58%), followed by female 64±51 ha-1 (36%) while bisexual were very rare (6%).Anthropogenic pressure like lopping for timber 28%, fuel wood 56%, debarking 11% and fencing 5% of Juniper tree equally treacherous at all populations. Leaf samples of the key species J. excelsa were collected for DNA isolation as no genetic analysis was ever carried out in this area. Owing to the existence of impurities of high magnitude of secondary metabolites, a novel protocol was generated to isolate pure DNA essentially for tree J. excelsa, which exhibited rich genetic resources and diversity. Six microsatellite primers were inspected and four showed considerable polymorphic amplifications. The percentage of polymorphic bands of populations was great with a variation of 8.8% as of maximum (61.8%) at Killi Shaban and minimum (53%) at Gunda. The polymorphism decreased with decrease in cover. The genetic diversity among populations also varied greatly, from 0.1012 - 0.2012 and their order in five villages were as Killi Shaban > Killi Tor Shore > Medadzai > Sarobai> Gunda. Results revealed a positive correlation between genetic diversity and annual mean precipitation, high and low temp and soil pH, and variance in these due to climate change might intimidate the genetic diversity and resources. The traditional healers are dwindling in number and traditional knowledge is threatened and might be exterminated in near future since younger generation is least interested in it. Therefore, it is preordained to preserve the indigenous knowledge. Geographical distance and density might have sound effects on genetic relationships, because genetic diversity improved even at short distance. Existing anthropogenic pressure on ecosystem can be curtailed by regulations implementations and financial assistance for sustainable resourse utilization to the unique Juniper ecosystem of Zarghoon.