Trypanosomosis is a vector-borne protozoan disease of animals and humans in sub-Saharan Africa. In Ethiopia, particularly the northwest region is affected by both tsetse and non-tsetse transmitted trypanosomosis. The aim of the present study was to determine the effects and compare differences in virulence of Trypanosoma vivax infection between tsetse and non-tsetse infested areas of northwest Ethiopia on the basis of serum biochemical values in Zebu cattle. Eighteen cattles purchased from trypanosome free area and aged between 9 and 12 months were assigned into three groups of six animals (Group TT=infected with T. vivax from tsetse infested area, Group NT=infected with T. vivax from non-tsetse infested area and Group C=non-infected control). For each experimental animal 3ml of blood from naturally infected cattle was inoculated intravenously at 106 trypanosomes/ml except the control. Blood sample was collected once a week for 8 consecutive weeks for analyzing serum biochemical values (glucose, total cholesterol, total protein, albumin, and enzymes including GOT, GPT and ALP) using a Humastar 80 clinical chemistry analyzer. Both T. vivax parasites caused an acute infection with parasites appearing in circulation on 6 and 12 days post-infection for NT and TT cattle, respectively. A significant reduction (P<0.001) in glucose levels was observed in infected groups compared with the control with mean values of 33.8±3.6mg/dl for TT, 34.3±3.6mg/dl for NT and 70.9±3.0mg/dl for control groups. A similar reduction was also seen in total cholesterol values (P=0.001) with 70.4±10.6mg/dl for TT and 78.0±10.6mg/dl for NT groups compared to 139.5±8.7mg/dl for the control group. No difference was observed for total serum protein between the three groups (P=0.260) whereas the mean albumin level was significantly (P<0.001) decreased (3.5±0.1g/dl and 2.9±0.1g/dl in TT and NT groups respectively) compared to that for control cattle (4.5±0.1g/dl). On the other hand, infected groups had higher ALP values compared to the control (P=0.007), with a mean value of 538.4±64.4IU/L, 564.9±64.4IU/L and 273.2±52.6IU/L for TT, NT and control cattle, respectively. In conclusion, the two T. vivax parasites caused significant biochemical changes indicative of pathological responses. However, there was no significant variation between the two parasites in initiating these changes despite the difference in the onset of parasitaemia.