In this study we report the usefulness of nested PCR for screening of the persistent B. microti infections in rodent hosts. Female BALB/c mice were inoculated with 100 mul of donor blood infected with B. microti. Infections were detected using microscopic examination of Giemsa-stained blood smears. To determine whether B. microti DNA was present in blood and/or spleen tissue, nested PCR was performed targeting a specific fragment of the gene encoding the 18S rRNA. Blood was sampled every 10 days post-infection (dpi) until day 30, after which mice were sampled every 30 days until the end of experiment at 360 dpi. The most extensive parasitaemia (39% of infected erythrocytes) was observed at 10 dpi. Between 20-60 dpi, less then 1% of infected erythrocytes were detected in blood smears, and from 90 dpi onwards, infected erythrocytes were no longer observed. B. microti DNA was successfully amplified from the blood of mice from 10 dpi until 180 dpi, as well as from spleens of infected mice at 10 and 20 dpi. The presented results show that nested PCR is the method of choice for monitoring infections of B. microti in the blood of rodent hosts, and could therefore be a tool for environmental monitoring of naturally infected rodents which are the predominant source of infection for tick vectors.