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The phylogeography of the filed vole (Microtus agrestis) in Eurasia was thoroughly examined using mitochondrial DNA (mtDNA) of the cytochrome b (cytb) gene. However, the former conclusions about genetic variability and the contact zone of eastern and western genetic lineages in Lithuania were based on the analysis of a very limited number of individuals. In the present study, we examined 74 M. agrestis individuals trapped in four sites in the eastern, northern, and western parts of the country using sequence analysis of cytb and D-loop. Totals of 25 new cytb haplotypes and 19 new D-loop haplotypes were identified for this species. Higher nucleotide diversity was observed for D-loop (π = 0.01147 ± 0.00070) as compared to cytb (π = 0.00694 ± 0.00039). The phylogenetic analysis based on both loci revealed the presence of two genetic lineages, i.e., the eastern and western ones, which were mixed in Lithuanian samples, with the exception of the Rusnė site in the west of the country. Only the western lineage was observed in this island population of M. agrestis; the sample differed in low genetic variability and genetic differentiation from other investigated samples. We found D-loop to be an appropriate locus for the evaluation of the genetic variability of M. agrestis. [ABSTRACT FROM AUTHOR]