ABSTRACT Blacklegged ticks, Ixodes scapularis Say, were collected from 27 sites in eight New York State counties from 2003 to 2006 to determine the prevalence and distribution of tick-borne pathogens in public-use areas over a 4-yr period. In total, 11,204 I. scapularis (3,300 nymphs and 7,904 adults) were individually analyzed using polymerase chain reaction to detect the presence of Borrelia burgdorferi (causative agent of Lyme disease), Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, causative agent of human granulocytic anaplasmosis), and Babesia microti (causative agent of human babesiosis). Overall prevalence of B. burgdorferi, A. phagocytophilum, and B. microti was 14.4, 6.5, and 2.7% in nymphs and 45.7, 12.3, and 2.5% in adult ticks, respectively. Rates varied geographically and temporally during the time period examined, and were related to measurements of tick density. Average rate ofpolymicrobial infection for nymphs and adults, respectively, was 1.5 and 8.5% overall, with 0.5 and 6.3% coinfection of B. burgdorferi and A. phagocytophilum, 1.0 and 1.5% B. burgdorferi and B. microti, and 0.05 and 0.6% A. phagocytophilum and B. microti. Thirty-three individual adult ticks from seven study sites in Westchester, Putnam, Dutchess, and Rockland counties tested positive for simultaneous infection with all three agents by multiplex polymerase chain reaction assay.