Tularemia, caused by Francisella tularensis (F. tularensis), is a zoonotic disease transmitted through contact with infected animals and contaminated environment. The disease has been reported from many countries of the world but no study has been done in Pakistan. In the current project, a total of 2280 soil samples representing 456 villages of eight districts of Punjab province were collected from way-points having human-animal interaction, processed for genomic extraction and tested through real time PCR for presence or absence of F. tularensis. Association of risk factors was determined from data such as gender and age of animals, plough method, irrigation system, fertilizer type used, availability of veterinary services, level of farmer education, physical and chemical composition of the soil. Moreover serum samples (n=707) collected randomly from goat (n = 200), sheep (n = 175), cattle (n = 179), and buffalo (n = 153) were analyzed for antibodies against F. tularensis by using enzyme-linked immunosorbent assay. Seventy four soil samples (3.24 percent) were found positive for F. tularensis. Phylogenetic analysis showed 100 percent similarity index with F. tularensis sub specie holarctica reported from other regions like USA, Sweden, Spain, Turkey and Germany. Presence of F. tularensis in soil showed negative association with increase in number of human density (0.7159; 0.3834- 0.2054). Prevalence of anti- Ft ELISA antibodies were significantly higher (p<0.05) in large ruminants (cattle and buffalo) as compared to small ruminants (goat and sheep). Age and gender- wise analyses showed non-significant differences (p>0.05) between small and large ruminants. Whereas, rain-irrigation system (2.96: 1.35- 6.48), lack of veterinary services (4.77:1.26-18.03) and use of organic fertilizer (5.3: 11.38- 20.39) have positive association with prevalence of anti- Ft ELISA antibodies in the serum. Sero-prevalence of F. tularensis in the animals has significant viiassociation with quantity of clay in soil (p<0.05). A conventional PCR based test has also been optimized for detection of F. tularensis using tul4 gene specific primers. Specificity of primer showed Ft detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/μL. Utilizing pET28a vector, a construct was prepared containing transformed tul4 gene (450bp) showing 100 percent sequence homology to query gene sequence. For manufacturing diagnostic assays especially in developing countries where availability of BSL-3 facilities and positive control reagents is an issue, provision of tul4 gene based constructs in vector can act as positive control and safe to use.