Punjab Urial is a wild sheep which is endemic to Northern Punjab, Pakistan. The species has been categorized vulnerable by IUCN Red List of Threatened Species. The Urial population has declined by 30% ove the last three generations. Its population is severely facing poaching & hunting stress so, working hard for their survival. Nematode infections may also compromise Urial survival, but little is known about Punjab Urial gastrointestinal nematodes. On the other hand, gender identification of wild ungulates is imperative and critical, particularly for juveniles. Accurate gender determination is important for studying ecology and conservation biology. Therefore, a simple molecular technique, that is precise with non-invasive sampling approaches such as faeces, would be valuable. This study focuses population estimation of Punjab Urial in Kalabagh Game Reserve District Mianwali, exploiting feacal samples as non-invasive source of identification. This study was designed for estimation of Urial population size, enumerate males and females present in the study area and to assess nematode infection. Feacal samples (n=215) were collected between April 2016 and September 2017. Those were subjected to identify as target species exploiting Urial specific primers (Urial_F/Urial_R). In this study, a novel DNA barcoding approach was developed using ITS-I as a target region, with a primer pair designed to amplify frequently reported nematode species for small ruminants. The novel primer pair was validated in silico and in vitro and subsequently used to determine the presence of nematodes in Punja Urial samples. DNA barcoding revealed a higher prevalence of Haemonchus contortus (73.91%), Trichuris ovis (16.30%) and Trichostrongylus axei (3.26%) in Punjab Urial. We genotyped the Urial samples using 12 sets of microsatellite markers (recommended by FAO) to see the genetic variation, population structure and demographic changes. Microsatellite analysis exhibited a high level of genetic variety in Urials in terms of expected and witnessed heterozygosity and allelic diversity. The population structure of Urial sheep, based solely on microsatellite variation using Bayesian clustering indicated three different clusters of the Punjab Urial occurring in Kalabagh Game Reserve. The AMOVA results indicated that the most of the genetic variation is based on the differences among the individuals. The results revealed that Urial population is facing inbreeding pressure and its ancestral effective population size has drastically reduced from 20,000-50,000 to about 1000 animals or less today. This reduction has occurred as a result of a bottleneck which occurred about 10,000 years ago. The results of bottleneck also indicated that cluster3 of Urial population has signature of bottleneck. In addition to this a set of molecular markers was developed exploiting the AMLx/y gene to assess gender of Punjab Urial population in KalaBagh, using faecal samples as the DNA source. In our study, among 92 Urial samples, 54 (58.69%) were identified as female samples, 34 (36.95%) were recognized as males while for remaining 4 (4.34%) samples there was no amplification, resulting in their gender not being able to be assessed. This study demonstrates that the novel DNA barcoding approach is a robust tool to detect nematode parasites from faecal samples of Punjab Urial. This method can detect nematode infections for surveillance and population conservation of the vulnerable Punjab Urial. This non-invasive sampling technique accurately identified gender and has importance in developing conservation application for Punjab Urials as well as equally applicable to other wild ungulates. Our finding suggests that Urials in the studied area are distributed in three cluster and cluster 3 has shown signs of inbreeding which is may be because of habitat isolation. One possible reason is: the three clusters are quite small and broadly dispersed into fairly accessible territory, and hence can be effortlessly vanished without any chance for the region to be naturally re-populated through dispersion.