A cross-sectional epidemiological survey was conducted to check the seroprevalence of 3 reportable equine diseases viz., equine infectious anemia (EIA), glanders, and piroplasmosis in 5 draught equine populated urban areas (Lahore, Gujranwala, Faisalabad, Multan and Bahawalpur) of Punjab. Assuming the expected prevalence to be 50 percent (unknown status) with confidence limits of 95% and a desired absolute precision of 5%, a total of 430 blood and sera (comprising 332 donkeys, 65 horses and 33 mules) along with relevant information were randomly collected. The samples were subjected to microscopic examination (thin and thick Giemsa stained smears), hematological analysis and commercial enzyme linked immunosorbant assay (ELISA). Sera were tested for EIA through ELISA (VMRD, Inc., USA), for glanders through rose Bengal plate agglutination test (RBT) and a commercial competitive ELISA (cELISA, VMRD, Inc., USA) was used for piroplasmosis screening. Positive and negative samples were identified on the basis of degree of agglutination (++ to ++++ considered positive) in case of RBT and as per instructions of the manufacturer regarding ELISAs. Data thus generated was analyzed by using epidemiological software tools (EpiinfoTM, WINPEPI, Survey Toolbox and IBM SPSS). Results: Giemsa stained smears were negative for blood parasite. None of the samples was positive for EIA, indicating towards possible disease free status or failure of the disease to mount immune response in sampled equine population. Seroprevalence of glanders was found to be 7.9% (n=34) with highest prevalence in Lahore (11.6%) followed by Bahawalpur (10.3%), Multan (7.3%), Faisalabad (6.7%) and Gujranwala (5.3%). Significantly different (P<0.05) seroprevalence was recorded in horses (16.9%) than mules (12.1%) and donkeys (5.7%). Seroprevalence recorded in young (< 5 years), adult (< 10 years) and old (> 10 years) equines was 7.9%, 9.2% and 5.2% respectively. Prevalence was significantly different (P<0.05) in males 5.7% and female 12.59% equine. Multivariable analysis indicated that female equines (OR = 2.3, 95% CI= 1.16–4.77) and and those sharing water sources or access to communal water troughs (OR =2.7, 95% CI=0.93-7.86) were more likely to be sero-positive for glanders. Seroprevalence of piroplasmosis was found to be 52.5% (T. equi=41.2% and B. caballi=21.6%; P<0.05) in the selected areas. Forty four (10.2%) sera were found positive for both piroplasms (mixed infection). Significantly higher (P<0.05) seroprevalence was recorded in Lahore (95.6%, n=66) than Faisalabad (64%, n=96) than Multan (52.9%, n=36), Gujranwala (50.7%, n=38) and Bahawalpur (50%, n=34). Seroprevalence of piroplasmosis was 69.2% in horses (T. equi=56.9%, n=37; B. caballi=24.6%, n=16) followed by 63.6% in mules (T. equi 48.5%, n=16; B. caballi 30.3%, n=10) and 48.2% in donkeys (37.3%, n=124). Adjusted analysis for possible confounding factors revealed that equines living alone or with equine cohorts (OR=1.30, 95%CI=0.84–2.01), with ticks infested equine cohorts (OR=1.19, 95%CI=0.65-2.18) whose owners did not practice tick control (OR=1.43, 95%CI=0.93-2.21) were more likely to be sero-positive for piroplasmosis. Hematological alterations observed in RBT (glanders) positive equines regarding white blood cell counts, red blood cell counts, hemoglobin concentrations and hematocrit in all there RBT positive equine species. Macrocytic hyperchromic type of anemia was observed in all RBT positive horses, mules and donkeys. Hematological analysis of piroplasmosis positive equines indicated decreased white blood cell counts, red blood cell counts PCV and hemoglobin concentration values in seropositive horses, donkeys and mules. Erythrocytic indices pointed towards microcytic hyperchromic type of anemia in piroplasmosis sero-positive equines. In conclusion, the high RBT based prevalence of equine glanders indicates towards possible endemic nature of this disease in Pakistan. Potential role of communal water sources and faulty management in the spread of equine glanders requires further investigations through carefully designed experimental studies. Seroprevalence of piroplasmosis was alarmingly high in the selected population which could be attributed to the faulty housing, lack of tick control practiced by owners and lacuna regarding knowledge among animal health professionals about the correct diagnosis and treatment of T. equi and B. caballi.