Data presented in this study focuses on the presence of anti-Borrelia antibodies in small mammals from Eastern Slovakia during 2000-2003. The total seropositivity observed was 18.78%in rodents. Amongst all species, the total seroprevalence in Apodemus flavicolis was the highest (20.87%), followed by Apodemus agrarius (19.58%) and Clethrionomys glareolus (11.11%). However, the prevalencein Apodemus flavicolis during the year 2000-2001 was higher (26.72%), which reduced to 10.60% in 2002-2003.To compare the year range of seroprevalence in other small mammals was not feasible due to the small sample number. Area-wise distribution of anti-Borrelia antibodies was even (18.75% to 20%) in this study, except in the Bot'any province (0%). This confirms the equal distribution of Borrelia spirochetes in the other 3 localities. Prevalence of anti-Borrelia antibodies during summer was significantly higher than during autumn and early spring. The overall study also reviews the importance of small mammals in Lyme disease ecology.