The present study was conducted to determine the seroprevalence of toxoplasmosis, hematological and serum biochemical changes in sheep naturally infected with Toxoplasma gondii and compared between T. gondii positive and negative sheep in Multan and Khanewal districts, Punjab (Pakistan). Blood samples were collected from 500 sheep and T. gondii antibodies were tested by latex agglutination test (LAT) and Enzyme Linked Immunosorbent Assay (ELISA). Information related to hosts and herds were recorded on questionnaire basis such as gender, age of animals, aborted or non-aborted sheep, cats association with herd, method of disposing aborted fetuses and herd size. The overall prevalence of toxoplasmosis in sheep was 33.6% by latex agglutination test (LAT) and 27.4% by ELISA (P-value = 0.033, OR = 1.341, 95 % CI = 1.023, 1.757). The area wise prevalence of toxoplasmosis was 34.02% and 33. 01 % by LAT (P-value = 0.813, OR = 1.031, 95 % CI = 0.802, 1.324) in Multan and Khanewal and 28.12 % and 26.41 % by ELISA (P-value = 0.672, OR = 1.065, 95 % CI = 0.796, 1.424) in Multan and Khanewal respectively. Difference was statistically non-significant (P>0.05). The seroprevalence of toxoplasmosis was compared between aborted and non-aborted sheep and according to these results infection rate was 51.66% and 31.10% in aborted and non-aborted sheep by LAT (P-value = 0.002, OR = 1.661, 95 % CI = 1.248, 2.211) and 41.66 % and 26.22% in aborted and non-aborted sheep by ELISA (P-value = 0.013, OR = 1.589, 95 % CI = 1.128, 2.238) respectively. Difference was statistically significant (P<0.05) by both techniques. The relationship between sex of sheep and toxoplasmosis showed that infection was 31.37% and 33.85 % by LAT (P-value = 0.722, OR = 0.927, 95 % CI = 0.605, 1.419) in males and females respectively. Similarly prevalence was 19.6 % and 28.28% by ELISA (P-value = 0.188, OR = 0.693, 95 % CI = 0.390, 1.232) in males and females respectively. The prevalence was higher in females than males and difference was statistically non-significant (P>0.05) by both techniques. The prevalence of T. gondii was compared between different age groups and according to these findings infection rate was higher in age group 60-73 months (64.5% and 38.7%) and lowest in age group 4-17 months (26.4% and 22.4%) by LAT (P-value = 0.001) and ELISA (P-value = 0.404). Difference was statistically significant (P<0.05) by LAT and non-significant (P>0.05) by ELISA. Risk factors associated with prevalence of toxoplasmosis in sheep were also studied and according to these results a large flock size, presence of cats, and the aborted fetuses openly left on ground were all statistically significant (P≤0.05) risk factors that were positively associated with prevalence of toxoplasmosis in sheep flocks. Hematological and serum biochemical parameters were studied between T. gondii positive and negative sheep and according to these results mean values of haemoglobin, glucose, cholesterol, AST, ALT and LDH were 9.5 ± 2.6 g/dL, 50.6 ± 11.3 mg/dL, 211.6 ± 79.6 mg/dL, 216.4 ± 144.4 U/L, 79.0 ± 64.1 U/L, 613.9 ± 297.2 U/L in T. gondii positive sheep by LAT and 9.9 ± 3.1 g/dL, 52.3 ± 11.3 mg/dL, 198.5 ± 73.6 mg/dL, 209.44 ± 629.3 U/L, 98.7 ± 228.7 U/L, 578.3 ± 292.9 U/L in T. gondii negative sheep respectively. Similarly mean values of haemoglobin, glucose, cholesterol, AST, ALT and LDH were 9.5 ± 2.5 g/dL, 50.6 ± 11.3 mg/dL, 214.6 ± 79.9 mg/dL, 217.3 ± 140.7 U/L, 77.8 ± 66.4 U/L, 606.8± 291.5 U/L in T. gondii positive sheep by ELISA and 9.9 ± 3.1 g/dL, 52.3 ± 11.2 mg/dL, 198.4± 73.8 mg/dL, 209.6 ± 605.1 U/L, 97.6 ± 219.8 U/L, 583.8 ± 295.7 U/L in T. gondii negative sheep respectively. Hemoglobin, glucose and ALT concentration was lower in T. gondii positive sheep as compared to T. gondii negative sheep. Cholesterol, AST and LDH concentration was higher in T. gondii positive sheep than T. gondii negative sheep. Haemoglobin, Glucose, AST, ALT and LDH values varied statistically non-significantly (P>0.05) between T. gondii positive and negative sheep whereas cholesterol level varied statistically significantly (P<0.05) between T. gondii positive and negative sheep. In conclusion, cholesterol level was found higher in sheep infected with toxoplasmosis which may lead to certain complications including coronary artery diseases, myocardial infarction, atherosclerosis, hypertension, hemorrhage and vascular thrombosis in infected sheep. T. gondii is a blood parasite which can cause abortion, fetal death, stillbirth, neonatal death in sheep which can lead toward economic losses such as reduction in fur, wool, meat, milk and other dairy products etc. The results indicated the overall high rates of seroprevalence of T. gondii in sheep that warranted the threat to public health especially farmers and abattoir workers via zoonotic transmission of toxoplasmosis. Keeping in view, the medical and veterinary importance of T. gondii it is recommended that human population (especially farmers and abattoir workers), other livestock species and stray cats present in study area must also be screened for the Toxoplasma infection in the public interest.