Infections of Babesia microti (Apicomplexa, Piroplasmida), a common erythroparasitic protozoon of Holarctic rodents, are not widely acknowledged in Poland. The presence of this parasite in various species of wild rodents has been well documented throughout the northern temperate zone of North America, Europe, and Eurasia. However, human babesiosis attributable to infection with B. microti has been reported only from the north-eastern and upper midwestern United States and Japan. We recently carried out an epizootiological survey investigating the prevalence of B. microti both in the tick Ixodes ricinus and in wild rodents in North-Eastern Poland. Blood samples were collected from a total of 483 animals comprising three species: Apodemus flavicollis, Microtus arvalis, and Microtus oeconomus trapped at Urwitałt near Mikołajki in the Mazury Lakes District. Questing adult I. ricinus ticks were collected in the study sites by blanket dragging of vegetation in heterogeneous, deciduous woodland, and, in addition, rodents were carefully examined for feeding larvae and nymphs. Altogether, B. microti was detected in 9 out of 1513 I. ricinus ticks (0.6%) examined by PCR. This included 163 adults (92 females and 71 males), 50 nymphs, and 1300 larvae 3%, 8%, and 0% of which were PCR-positive, respectively. Of 85 A. flavicollis, 374 M. arvalis and 24 M. oeconomus, 1%, 12.8%, and 42% were parasitaemic, respectively, as determined by microscopic examination of blood smears stained with Giemsa. B. microti DNA, extracted from 53 M. arvalis and 5 M. oeconomus and examined by nested PCR, targeting a piroplasm-specific portion of the 18S ribosomal DNA, revealed 72% and 40%, respectively, to be PCR positive. Sequence analysis showed that all PCR-positive samples had rDNA sequences identical (100% homology) to that of the Munich B. microti strain isolated from Mus musculus. The results of this study indicate that the B. microti commonly encountered among Microtus spp. rodents is probably not a zoonotic strain and, therefore, that it is most unlikely to represent a risk to public health in the Mazury Lakes District of North-Eastern Poland.