Intensive small mammal trapping was conducted in 12 counties in New York state during 1998–2000 to investigate the prevalence and site specificity of the Lyme disease spirochete, Borrelia burgdorferi in, and presence of the blacklegged tick, Ixodes scapularis Say on, the wild mice Peromyscus leucopus Rafinesque and Peromyscus maniculatus Wagner and other small mammal species. Previously captured mice (1992–1997) from throughout New York state also were recruited into the study, providing a total of 3,664 Peromyscus from 107 sites in 31 counties. Infection with B. burgdorferi was determined by polymerase chain reaction testing of ear tissue, and rates were determined by species, counties, and regions of the state. B. burgdorferi was detected in 10 small mammal species captured during 1998–2000. Peromyscus captured from Dutchess County in the lower Hudson Valley had the highest infection rate of 21%. The next highest infection rates were in counties within the Capital District: Albany (18%), Rensselaer (17%), and Columbia (13%). From 4,792 small animals examined, we recovered 2,073 ticks representing six species from 414 individuals of 12 mammal species, including 1,839 I. scapularis collected from 315 Peromyscus trapped in five counties. I. scapularis were most often collected from animals trapped in Albany, Rensselear, and Dutchess counties. We used protein electrophoresis of salivary amylase to distinguish between P. leucopus and P. maniculatus species. I. scapularis burdens were 5.7 ticks per P. leucopus and 14.3 ticks per P. maniculatus.