This is the first study to document the genetic diversity of the white-tailed deer population on St. John, US Virgin Islands. The island population was founded by a small number of animals, has very limited hunting or predation, and recently experienced a reduction in size following an extended drought in 2015. DNA samples were collected from hair from 23 anesthetized adult deer (13 males, 10 females) ranging in age from 1 to 8 years (3.36 ± 1.9 years) and also from fecal DNA samples, for a total of 42 individuals analyzed for genetic diversity. The St. John deer data set averaged 4.19 alleles per marker and demonstrates the second lowest number of alleles (A) when compared to other populations of Odocoileus virginianus (4.19). Heterozygosity was similar to the other studies (0.54) with little evidence of inbreeding. To explain the level of heterozygosity and level of inbreeding within the St. John population, three hypotheses are proposed, including the effect of intrinsic biological traits within the population, a recent infusion of highly heterogeneous loci from North American populations, and a consistent level of immigration from a nearby island. Additional work is needed to further understand the genetic history of the St. John and regional deer populations.