The present study was designed to compare the sensitivity and specificity of different parasitological and molecular techniques for the detection of Trypanosoma sp. during different seasonsof year 2013 in equines and camels of Dera Ghazi Khan District and to determine the risk factors associated with the spread of trypanosomiasis. The objective of this project was to demonstrate the effect of Trypanosoma sp. on complete blood count (CBC) and selected serum biochemical parameters in camels, horses and donkeys from Southern Punjab (Pakistan). A total of 858 blood samples (291 camels, 284 horses and 283 donkeys) were collected and were subjected to blood smear examination, hematocrit centrifugation technique and Polymerase Chain Reaction (PCR) (by amplifying kinetoplast maxicircle DNA) to determine the prevalence of trypanosomiasis in collected samples. Blood samples were also analyzed for complete blood count and selected serum biochemical parameters (Total Protein, Creatinine, Alanine Transferase (ALT), Aspartate Transferase (AST) and Triglycerides). Out of 291 camel blood samples, 28 (9.62%) generated a 164 bp DNA fragment specific for Trypanosoma sp. Only 6 blood samples from camels (2.1%) were found positive through microscopic examination while 13 (4.46%) were positive for Microhematocrit centrifugation technique. Out of 284 blood samples from horses 16 (5.63%) samples were found Trypanosoma sp. positive by PCR. Only 5 blood samples (1.8%) were found positive through microscopic examination while 9 (3.17%) blood samples from horses were positive for Microhematocrit centrifugation technique. Out of 283 blood samples of donkeys, 19 (6.71%) samples amplified a 164 bp DNA fragment specific for Trypanosoma sp. while only 7 blood samples (2.5%) were found positive through microscopic examination and 9 (3.18%) were found positive for Microhematocrit centrifugation technique. Seasonal prevalence through PCR was 6.9% (5/72) in spring, 13.7% (10/73) in summer, 9.7% (7/72) in autumn and 8.1% (6/74) in winter in camels. Seasonal prevalence through PCR was 4.2% (3/72) in spring, 5.9% (4/68) in summer, 7.04% (5/71) in autumn and 5.48% (4/73) in winter in horses. In donkeys, seasonal prevalence through PCR was 4.76% (3/65) in spring, 8.22% (6/73) in summer, 8.45% (6/71) in autumn and 5.41% (4/74) in winter. No significant association (P˃0.05) of disease was observed with age of all studied animals. Trypanosomiasis was also found not to be associated (P˃0.05) with the gender of equines and camels during present study. A significant increase was detected in white blood cells (WBC) (P˂0.001), neutrophils(P˂0.004) and ALT(P˂0.028) in Trypanosoma sp. positive as compared to the Trypanosoma sp. negative blood samples while a significant decrease in red blood cells (RBC) (P˂0.001), hemoglobin (P˂0.001), lymphocytes (P˂0.003) and packed cell volume (P˂0.001) was observed in blood samples of camels. In horse blood samples, there was also significant increase in WBC (P˂0.001), neutrophils (P˂0.001) and ALT (P˂0.032) and a significant decrease (P˂0.001) in RBC, hemoglobin (P˂0.001), lymphocytes (P˂0.021) and packed cell volume was observed in parasite positive as compared to the parasite negative blood samples. In donkeys, a significant increase was detected in WBC (P˂0.002), neutrophils (P˂0.001) and ALT (P˂0.019) in Trypanosoma sp. positive as compared to the Trypanosoma sp. negative blood samples while a significant decrease in RBC (P˂0.001), hemoglobin (P˂0.003), lymphocytes (P˂0.001) and packed cell volume (P˂0.003) was observed. In conclusion, PCR was found more reliable and sensitive technique than the blood smear examination and microhematocrit centrifugation technique for Trypanosoma sp. detection in blood samples of equines and camels and it is recommended to be used for Trypanosoma sp. detection in epidemiological surveys and control policies in livestock sector to minimize economic losses.