Islands have a long history in species conservation, as they often have fewer predators and competitors than mainland sites. In some instances, islands have been used as breeding colonies to support mainland translocations. In the past decade, 2 islands in New England have been selected to establish island breeding colonies of New England cottontail (Sylvilagus transitionalis), a species of conservation concern. The 2 islands, Patience Island and Nomans Land Island National Wildlife Refuge (NWR), were initiated in 2012 and 2019, respectively. Our main objective was to monitor the genetic diversity and population size response of the removal of New England cottontails from Patience Island for translocation to mainland sites. This allowed us to test theoretical predictions about the species' change in genetic diversity over time on an island system. As expected, the mitochondrial DNA haplotype diversity of New England cottontail decreased at a faster rate than their nuclear DNA heterozygosity, the latter remaining stable for over a decade. We also radio‐collared founders released on both islands to estimate their individual survival, which decreased as additional founders were released on Patience Island. We tested the influence of outcrossing parents from different populations on the survival of their released offspring on Patience Island, which had similar survival as offspring with parents from the same population. The survival of Nomans Land Island NWR founders was higher than that of Patience Island founders, likely because of the lack of mammalian predators on Nomans Land Island NWR. Islands have played a critical role in supporting New England cottontail conservation and, in the right circumstances, islands could be used to support the conservation of other lagomorphs of conservation concern. However, careful consideration is needed before an animal is introduced to an island to reduce the possible negative impacts to the island's fauna and flora and maximize the potential success of the island population in supporting the focal species' conservation.