Two Hawaiian endemic yeast species, Metschnikowia hawaiiensis and Metschnikowia hamakuensis, were examined by means of multilocus characterization. In spite of their narrow range of distribution, restricted to the island of Hawai‘i, both species were found to be polymorphic at several loci. Alleles of different loci were distributed independently within local populations, confirming that sexual reproduction prevails among these facultatively asexual organisms. No alleles were shared across species, confirming their reproductive isolation. Although the sample size for the northern species, M. hamakuensis, is much less (N = 7) than that for its southern relative, M. hawaiiensis (N = 161), their genetic diversity is comparable. Genetic differentiation was detected in M. hawaiiensis populations at both regional (ca. 30–40 km) and local (ca. 1 km) scales. A single isolate recovered in a separate locality exhibited considerable allelic divergence from others, indicating that genetic isolation can occur over relatively short distances and suggesting a first step towards cessation of gene flow, which is required for speciation.