Economically, Pakistan is an agricultural country as the agriculture sector offers employment to 42.3% of the country?s citizens involved with any kind of labor for their livelihood, 19.5% of its gross domestic product (GDP) comes from the agriculture sector apart from the raw materials for many other value-added sectors contributing to huge quantities of added indirect income; livestock sector as for instance, adding 58.33% to the worth of agriculture besides contributing 11.39% directly to GDP. Population of livestock are cattle 44.4, Buffalo 37.7, Sheep 30.1, goat 72.2, Camel 1.1, Horses 0.4, Asses 5.2, Mules 0.2 millions. Asses population increased from 4.9 to 5.2 million while population of Camel, Horses and mules remained unchanged since last year. Horses along with mules and donkeys facing many managerial, nutritional, noninfectious and infectious diseases which decreases production and performance, out of which infectious diseases such as Glanders is life threatening and pose zoonotic potential to human beings specially care takers, veterinarian and public health. To monitor threat due to these asymptomatic carriers to animals and human beings and from places having decreases frequency of disease Glanders in equines, it is unavoidable to develop and optimize a highly specific and sensitive molecular test such as PCR, required to decrease the number of results which are false negative and false positive findings besides being fast and accurate. 200 nasal and blood samples of working equines (horses, mules and donkeys) were collected from the Bahawalpur, Faisalabad, Gujranwala, Lahore and Multan at the sites of Brook hospital. All the samples were collected and transported to the WTO Quality Control Laboratory of UVAS, Lahore keeping in account the all microbiological hygienic and hazardous measures according to OIE protocols. All the animals were injected mallein PPD intra-dermally into the lower eyelid with the result noted after first and second day. Blood of animals were used for DNA extraction by commercially available DNA extraction kit. The extracted DNA were subjected to Gel Electrophoresis for integrity of the extracted DNA and then used further for amplification of target region of the DNA. The extracted DNA was used for the amplification of Flip IS407A gene region using the specific primers. Products of the PCR were visualized with UV light after electrophoresis of the gel and B. mallei was detected in only positive control. The study found high prevalence of the disease and existence of active nasal shedders in equines. The findings implicate importance of targeted surveillance in glanders suspected equines.