Brucellosis is endemic in many livestock worldwide especially developing countries. The aims of this study were to estimate the seroprevalence of bovine brucellosis and risk factors associated with the seropositivity in rural and peri-urban buffaloes and cattle populations of Sindh. Firstly, a cross sectional study was conducted to estimate the seroprevalence of bovine brucellosis in cattle and buffaloes of Sindh province, Pakistan. Serum samples (2600) were tested using Rose Bengal Plate Test. The overall seroprevalence of brucellosis in Sindh province was 13.96% (95% C.I.; 11.55 - 16.37). Of the 917 herds tested, 232 or 25.30% herds (95%C.I.; 22.51-28.24) were positive for brucellosis. The adult animals were 2.05 (95% C.I.; 1.14-3.68, P= 0.02) times more likely to test positive for brucellosis. The animals in a peri-urban dairy production system were 2.07 times (95%C.I.; 1.09-3.90, P = 0.03) times more likely have brucellosis. The species or sex of animal did not appear to affect the risk of seropositivity in cattle or buffalo in this population. Secondly, a cross sectional survey was conducted to understand the structure and composition of farms, animal husbandry and management practices in peri-urban dairy colonies in Karachi and farmers’ awareness of zoonoses. The mean herd size was 93.58 animals and 88.01% of these animals were female buffaloes. Of 326 farms surveyed, only 37.42% were able to associate animals with transmission of diseases in human. The characteristics of peri-urban dairy farms in Karachi are discussed. Thirdly, the value of FTA® cards in detecting the Brucella DNA in milk samples was estimated by determining the detection limits of genus specific ERI PCR assay for FTA® cards and comparing the PCR results from whole sediments taken from culturing pooled milk samples with taking sediment on FTA® cards. The detection limits of this method ranged from 6.6 x 103 cfu/ml for B. abortus to 7.17 x 106 cfu/ml for B. suis. Assuming the results of ERI PCR for the whole sediment as gold standard (method 1), the method using sediment on FTA® cards as test samples (method 2) showed a diagnostic sensitivity of 81.44% (95% C.I.; 75.54-87.33) but a poor diagnostic specificity of 42.86% (95%C.I.; 16.95-68.78). The kappa value, κ, was 0.14 (p = 0.02) demonstrating a poor agreement between the two methods. Lastly, 181 bulk milk samples were used to estimate the herd level prevalence of bovine brucellosis in Landhi dairy colony, Karachi. The ERI PCR was used to test these samples. The herd prevalence was estimated as 92.26% (95% C.I.; 88.34-96.19). For each level (50 animals) increase in herd size, the risk of herd being brucellosis positive increases by 2.38 times. The herds that have a male animal for breeding are 0.09 times less likely to have brucellosis. The history of abortion, presence of small ruminants or the regions of animal purchase don’t appear to have any association with the risk of brucellosis at a herd level in this population at LDC, Karachi. A high seroprevalence of bovine brucellosis in livestock population in Sindh and a very high herd level prevalence in peri-urban dairy farms in particular poses a serious threat to the public health and livestock production in Sindh, Pakistan.