Lyme disease is the most common vector-borne illness in the United States. The most competent reservoir host for the infectious agent is the white-footed mouse (Peromyscus leucopus) and the vector that transmits the Lyme pathogen (Borrelia burgdorferi) to humans is the black-legged (Ixodes scapularis). To assess prevalence of B. burgdorferi within peri-urban and rural counties in the Southern Tier of New York State, ticks were collected where people were perambulating in varying microecologies in fragmented ecospaces. Collection proceeded by dragging a corduroy cloth over foliage and leaf litter and ticks attached to the cloth were placed in 70% ethanol and brought to the laboratory for pathogen analysis. Species, life cycle stage and sex of each tick were identified microscopically and DNA extracted and purified. The presence of B. burgdorferi was assessed by ospC PCR amplification and positive PCR products were Sanger sequenced. We collected and tested more than 1500 black-legged ticks for the presence of B. burgdorferi between October 2012 and November 2014 and found infectivity rates of 39.8% and 30.7% within Broome and Chenango counties, respectively. The density of infected ticks within Broome County was 1.9 per 1000m2. Our findings suggest that areas of high human activity with fragmented ecospaces within built environments such as college campuses, playgrounds, parks, jogging trails, and other recreational areas pose a significant risk of contracting Lyme disease. Human activities often perpetuate and increase the transmission of Lyme disease by enhancing reservoir and vector prevalence in these areas. We conclude that the Southern Tier of New York State may be on the cusp of a surge in incidence of Lyme disease over the next 5 years, likely moving slowly from east of the Hudson River westward into the Upper Susquehanna River Basin of New York State.