An understanding of genetic diversity and population structure is important to the conservation of declining species within fragmented habitats. These issues become critical for small, isolated populations, in which stochasticity is a main driver of genetic change and possibly of population extinction. In eastern New York and New England the endemic New England cottontail has declined due to habitat loss and fragmentation. As a species that exhibits metapopulation dynamics, habitat fragmentation can have profound implications for its persistence. We examined genetic diversity, population structure, and effective population size (Nₑ) of New England cottontails in New York, a purported remnant stronghold for the species. We amplified ten microsatellite loci from tissues collected from live-captures and from fecal pellets. We investigated potential hierarchical population structuring using programs STRUCTURE and BAPS. STRUCTURE identified four hierarchical tiers consisting of nine clusters, and BAPS clustering was highly consistent with that given by STRUCTURE. Most populations displayed significant genetic differentiation (FST = 0.04–0.34) and little to no evidence of ongoing connectivity. Low genetic diversity was observed based on allelic richness (2.2–3.0), and all populations had critically low effective population sizes (Nₑ; 2.7–57.1). Observed trends in population subdivision, genetic diversity, and Nₑ were consistent with reported trends in the state-endangered Maine-New Hampshire populations, and not indicative of a genetic stronghold within New York. Instead, the small and isolated populations observed here imply a breakdown in metapopulation functionality indicative of conditions faced by the species range-wide and an immediate need for human intervention to restore connectivity and rebuild populations.