Genetic diversity is critical to a population’s ability to overcome gradual environment change. Large-bodied wildlife existing in regions with relatively high human population density are vulnerable to isolation-induced genetic drift, population bottlenecks, and loss of genetic diversity. Moose (Alces americanus americanus) in eastern North America have a complex history of drastic population changes. Current and potential threats to moose populations in this region could be exacerbated by loss of genetic diversity and connectivity among subpopulations. Existing genetic diversity, gene flow, and population clustering and fragmentation of eastern North American moose are not well quantified, while physical and anthropogenic barriers to population connectivity already exist. Here, single nucleotide polymorphism (SNP) genotyping of 507 moose spanning five northeastern U.S. states and one southeastern Canadian province indicated low diversity, with a high proportion of the genomes sharing identity-by-state, with no consistent evidence of non-random mating. Gene flow estimates indicated bidirectionality between all pairs of sampled areas, with magnitudes reflecting clustering and differentiation patterns. A Discriminant Analysis of Principal Components analysis indicated that these genotypic data were best described with four clusters and indicated connectivity across the Saint Lawrence River and Seaway, a potential physical barrier to gene flow. Tests for genetic differentiation indicated restricted gene flow between populations across the Saint Lawrence River and Seaway, and between many sampled areas facing expanding human activity. These results document current genetic variation and connectivity of moose populations in eastern North America, highlight potential challenges to current population connectivity, and identify areas for future research and conservation.