In 2004, we collected 211 specimens of European field mice (genus Apodemus; subgenus Sylvaemus) from 16 localities in northern, western, central, and southern Ukraine, including the Crimean Peninsula. We used cytochrome-b mitochondrial DNA sequence data to investigate species diversity, distributional patterns, and taxonomy of Apodemus in Ukraine. Sequence data proved useful for species-level discrimination, because the 11 species examined were defined by low levels of intraspecific variation (mean range, 0.00–3.35%) and by high levels of interspecific variation (mean range, 5.37–18.9%). We identified the 211 specimens to 1 of 4 species: A. flavicollis (n = 121), A. sylvaticus (n = 34), A. uralensis (n = 16), and A. witherbyi (n = 40). Although all 4 species are known from Ukraine, this study provides new information about the ranges and population identity for these species in southeastern Europe. For example, our analyses substantiate the marked genetic structure reported for A. sylvaticus and document the presence of northern and southern continental lineages of this species in Ukraine that are hypothesized to have been isolated from each other in glacial refugia approximately 1.5 million years ago (>5% genetic divergence). Banks of the Dneiper River in northern Ukraine represent an area of secondary contact for the 2 lineages. Additionally, although application of A. witherbyi as a species-level name is debatable, examination of our genetic and morphological data supports its validity and priority over junior synonyms A. hermonensis and A. iconicus.