Milk is generally considered as highly nutritious and useful food for all age groups as well as inexpensive and widely available. However, its quality and microbial content are the features which have to be observed. The quality of milk depends on its biochemical composition and hygienic conditions during the collection of milk and its distribution. Milk itself acts as best medium for enhancing the growth and development of different types of microbes because of its composition and presence of water in large quantity. Milk sold in Bahawalpur district was suspected to be contaminated because of repeated outbreaks of gastroenteritis. In this context, the research was designed to rule out molecular detection and quantitative analysis of Escherichia coli (E. coli) in the milk samples obtained from various tehsils of Bahawalpur district. E. coli were considered as the most prevalent bacteria of milk and could be the most probable microbe causing this disease. In this research, 100 raw milk samples were gathered in a way that 10 samples each were collected from Jamalpur, Hasilpur, Khairpur, Qaimpur, Lal Sohanra, Lal Sohanra National Park, Yazman Mandi, Head Rajkan, Ahmedpur East and Uch Sharif. Then the samples of milk were cultured on different culture media for bacterial segregation. Identification of bacterial specie was done through gram?s staining and properties of bacterial culture on different selective media. Biochemical tests were also performed which include catalase and coagulase tests. Final identification was performed through PCR and resolution of PCR products by gel electrophoresis. Antibiotic sensitivity test was also performed so as to confirm the susceptibility of E. coli regarding multiple antibiotics. Out of 10 samples collected each from Jamalpur, Hasilpur, Khairpur, Qaimpur, Badar Sher, Lal Sohanra National Park, Yazman Mandi, Head Rajkan, Ahmedpur East and Uch Sharif, results had shown the presence of E.coli in 4(40%), 6(60%), 3(30%), 2(20%), 4(40%), 5(50%), 3(30%), 4(40%), 6(60%) and 5(50%) samples respectively. E. coli isolates were amplified by PCR based on 16S rRNA gene. Results of antibiotic sensitivity test revealed that E. coli isolates had shown resistance to amoxicillin (85%) and erythromycin (72%). However, they were found to be affected by azithromycin (53%), ciprofloxacin (86%), gentamicin (86%), norfloxacin (80%) and streptomycin (66%). Resistant pattern in relation to broad spectrum antibiotic (i.e., amoxycillin) points to a situation which should be considered carefully and suggests that indiscriminate use of antibiotics for precautionary or therapeutic purposes should be avoided as it could be the cause of increasing antimicrobial resistance.