Brucella species are cause of abortions and other reproductive disorders in animals and human beings. In this project, prevalence and distribution of soil borne Brucella species in nine districts of Punjab including Lahore, Faisalabad, Sheikhupura, Sargodha, D.G. Khan, Chakwal, Sahiwal, Gujranwala and Attock were determined. By using grid based sampling strategy, soil samples (n= 1280) were collected from villages (n=256, 5 samples/village) of the nine districts. Brucella genus specific DNA (bcsp31) was detected using Real Time PCR and prevalence was 2.11%. Out of five species, only two species B. abortus and B. melitensis were detected through conventioneal multiplex PCR. Prevalence of soil borne species specific DNA of B. abortus and B. melitensis was 1.8% and 0.31%, respectively. Out of nine, three districts were positive for Brucella species specific DNA. All districts showed a significant result for detecting Brucella species (χ2 =54.505, df=8, p<0.05, 95% CI). In conclusion, distribution pattern of the soil borne Brucella species in three districts of Punjab puts both human and animal population at a high risk of exposure. Further studies are required to explore molecular diversity of the pathogens together with sero-conversion in animals and humans.