Background: Trypanosoma spp. is a flagellated unicellular protozoan parasite which caused a disease known as trypanosomiasis in various animals among many areas worldwide resulting in severe economic losses due to morbidities and mortalities. This study was conducted to estimate the prevalence of Trypanosoma spp. in sheep using the molecular assay with detection the impact of infection on various risk factors and blood markers. Methods: Totally, 341 sheep were selected from different areas in Wasit province (Iraq) during June to September (2023) and subjected to draining the venous blood that tested molecularly by the conventional polymerase chain reaction (PCR). Status of different hematological, biochemical and mineral markers were further investigated. Result: Targeting 16S sRNA gene, 7.92% animals were positively infected with trypanosomiasis. Relation to risk factors, positive infection and risk were significantly higher in females than males and in sheep aged greater than 1-3 years than others. Hematological findings reported a significant reduction in RBCs and Hb; while biochemically, significant elevation was reported in concentration of urea. Regarding various minerals, insignificant alteration was seen in infected sheep when compared to healthy ones.