African animal trypanosomosis, transmitted cyclically by tsetse flies or mechanically by other biting flies, causes serious health problems in livestock. Although tsetse infestations have been observed in Blue Nile State in Sudan, tsetse was eradicated in West Kordofan in 1962, and no further studies have been carried out. Accordingly, in this study, we investigated the prevalence of trypanosomosis in cattle, sheep, and goats in Blue Nile and West Kordofan States, Sudan. This cross-sectional study was conducted using 70 cattle, 62 sheep, and 116 goats, and the microhematocrit centrifugation technique was used as a parasitological test. KIN-multispecies polymerase chain reaction (PCR) was used to detect Trypanozoon sp., Trypanosoma congolense, and T. vivax; RoTat 1.2 variable surface glycoprotein-specific PCR was used to detect T. evansi; and TviCatL PCR was used to specifically detect T. vivax. The seroprevalence of trypanosomosis was assessed using card agglutination tests CATT/ T. evansi. The parasitological prevalence rates were 4% (3/70) in cattle, 2% (1/62) in sheep, and 4% (5/116) in goats. The molecular prevalence rates of T. vivax, the most prevalent parasite, were 99% (69/70) in cattle, 98% (61/62) in sheep, and 84% (98/116) in goats. Trypanozoon (T. evansi or T. brucie) rates were 30% (21/70) in cattle, 32% (20/62) in sheep, and 12% (14/116) in goats. Among Trypanozoon-positive isolates, T. evansi was confirmed in 24% (5/21) of cattle, 70% (14/20) of sheep, and 86% (12/14) of goats. Finally, T. congolense was recorded only in cattle in Blue Nile State, with a prevalence of 14% (10/70). The seroprevalence rates of CATT/T. evansi were 46% (32/70) in cattle, 45% (28/62) in sheep, and 14% (16/116) in goats. Thus, we confirmed molecularly, for the first time, the presence of Trypanozoon, particularly T. evansi and T. vivax, in sheep and goats in Sudan. Our results show that sheep and goats could be an important reservoir for trypanosomes, potentially leading to the spread of the disease to the northern parts of the country following the movement of these animals. These findings provide important insights into the epidemiology of the disease and could affect the establishment of control strategies against trypanosomosis in Sudan.