We report here the first molecular evidence for the occurrence of Aricia artaxerxes (Fabricius, 1793) (Lepidoptera: Lycaenidae) in the Czech Republic. In Central Europe, this species may co-occur with its more common sibling, Aricia agestis (Denis & Schiffermüller, 1775). We sequenced the cytochrome c oxidase subunit 1 of darkly-coloured, putative A. artaxerxes specimens in the Czech Republic. We confirmed A. artaxerxes only from a limestone area in South Bohemia (Vyšenské kopce National Nature Reserve), which is probably the only locality of the species in the Czech Republic. This area is located at ca. 550 m A.S.L., showing that the elevation overlap with A. agestis could be high in Central Europe. Other surveyed individuals were confirmed as A. agestis, with a minimum p-distance of 1.98% between the two species. The South Bohemian area of occurrence is probably highly isolated (approx. 190 km) from localities of the species in neighbouring countries, highlighting the conservation importance of the A. artaxerxes population and of the insular calcareous areas in the Šumava Mountains foothills. We used database sequences of A. artaxerxes to place the Czech population into a wider phylogeographic context. The Czech population is monomorphic, consisting of a single haplotype, which is present from Scandinavia through Germany to Central Asia.