Trypanosomosis remains is a major endemic protozoal disease of mammalian livestock in tropical and subtropical areas with attendant economic losses, but epidemiological surveillance and diagnosis has remained based on relatively inefficient parasitological techniques. A molecular epidemiological survey using Polymerase Chain Reaction (PCR) based on species-specific primers of pathogenic trypanosomes (Trypanosoma vivax, T. congolense, T. brucei and T. evansi) was carried out in 4 major Nigerian cattle breeds. Of the 411 animals sampled, 61.1% tested positive by PCR for at least one of the four trypanosomes. T. vivax and T. congolense were the predominant trypanosomes detected by PCR respectively. There were no significant differences (p<0.05) in trypanosomal infection rates by sex, age and breed. However, cattle from the West had higher infection rates of trypanosomes than cattle from the North (p = 0.046). Mean packed cell volume (PCV) was not different between trypanosoma infected cattle and non-infected cattle, but was significantly different between infected cattle with poor versus good body conditions as well as between those cattle infected and non-infected by T. vivax. Molecular analysis revealed the presence of two variants of T. congolense (T. congolense savannah and T. congolense forest type) for the first time in Nigeria and demonstrated that bovine trypanosomosis is more prevalent in the West, and highest in White Fulani cattle breed compared to the North and other breeds of cattle in Nigeria. Therefore, extensive epidemiological survey could be carried out using PCR to detect bovine trypanosomes that are pathogenic to both animal and humans in Nigeria.