The distribution and spread of the tundra vole (Alexandromys oeconomus) in Lithuania have been documented over the last 70 years, but the genetic diversity of the species has not been studied. In this study, we examined A. oeconomus trapped in three sites in northern and western Lithuania using mtDNA sequence analysis of the cytb and control region. The western and northern sites are separated by anthropogenic landscape barriers. The western site is subject to regular spring flooding. Phylogenetic analyses of the studied individuals placed them in the Central European phylogroup, suggesting that Lithuanian A. oeconomus originated from northeastern Poland. In Lithuania, the genetic diversity of A. oeconomus at both mtDNA loci was relatively low (Hd < 0.6, π < 0.002) compared to that found in other European samples (Hd = 0.833–0.958; π = 0.00402–0.01552). Individuals analyzed in Lithuania were genetically different from samples collected in Poland and Northern Europe (ΦST > 0.15, p < 0.05). The genetic divergence between the western and northern samples of A. oeconomus in Lithuania, together with the low genetic variability among the voles studied, provides new insights into the phylogeography of the species and the influence of barriers on the colonization of the country.