BACKGROUND: Animal African Trypanosomiasis (AAT) is one of the most economically important diseases affecting livestock productivity in sub-Saharan Africa. The disease is caused by a broad range of Trypanosoma spp., infecting both wild and domesticated animals through cyclical and mechanical transmission. This study aimed to characterize trypanosomes present in cattle at regular intervals over two years in an AAT endemic and a non-endemic region of Ghana. METHODOLOGY/PRINCIPAL FINDINGS: Groups of cattle at Accra and Adidome were selected based on their geographical location, tsetse fly density, prevalence of trypanosomiasis and the breed of cattle available. Blood for DNA extraction was collected at approximately four to five-week intervals over a two-year period. Trypanosome DNA were detected by a sensitive nested PCR targeting the tubulin gene array and massively parallel sequencing of barcoded amplicons. Analysis of the data was a semi-quantitative estimation of infection levels using read counts obtained from the sequencing as a proxy for infection levels. Majority of the cattle were infected with multiple species most of the time [190/259 (73%) at Adidome and 191/324 (59%) at Accra], with T. vivax being the most abundant. The level of infection and in particular T. vivax, was higher in Adidome, the location with a high density of tsetse flies. The infection level varied over the time course, the timings of this variation were not consistent and in Adidome it appeared to be independent of prophylactic treatment for trypanosome infection. Effect of gender or breed on infection levels was insignificant. CONCLUSIONS/SIGNIFICANCE: Most cattle were infected with low levels of several trypanosome species at both study sites, with T. vivax being the most abundant. The measurements of infection over time provided insight to the importance of the approach in identifying cattle that could suppress trypanosome infection over an extended time and may serve as reservoir.