Abstract Biscutella laevigata L. is known as a Tl hyperaccumulator. In Poland Biscutella laevigata occurs in the Tatra Mts (Western Carpathians) and on the calamine waste heap in Bolesław near Olkusz (Silesian Upland). The purpose of this work was to evaluate whether plants of both populations were able to accumulate an elevated amount of thallium in their tissues. The plants were cultivated in calamine soil in a glasshouse for a season and studied at different ages – from 2-week-old seedlings to 10-month-old adults. Additionally, the plants were grown for ten weeks in calamine soil with EDTA to enhance Tl bioavailability. The total content of Tl in plant tissues after digestion was determined by ICP-MS, whereas its distribution in leaves was studied by LA-ICP-MS. Of the total content of Tl in the soil in the range of (15.2–66.7) mg∙kg−1d.m., only (1.1–2.1) mg∙kg−1d.m. was present in a bioavailable form. The mean content in all the plants grown on the soil without EDTA was 98.5 mg∙kg−1d.m. The largest content was found in leaves – 164.9 mg∙kg−1d.m. (max. 588.2 mg∙kg−1d.m.). In the case of plants grown on the soil enriched with EDTA, the mean content in plants increased to 108.9 mg∙kg−1d.m., max. in leaves – 138.4 mg∙kg−1d.m. (max. 1100 mg∙kg−1d.m.). The translocation factor was 6.1 in the soil and 2.2 in the soil with EDTA; the bioconcentration factor amounted to 10.9 and 5.8, respectively. The plants from both populations did not contain a Tl amount clearly indicating hyperaccumulation (100–500 mg∙kg−1d.m.), however, high (>1) translocation and bioconcentration factors suggest such an ability. It is a characteristic species-wide trait; B. laevigata L. is a facultative Tl hyperaccumulator. The largest Tl amount was located at the leaf base, the smallest at its top. Thallium also occurred in trichomes, which was presented for the first time; in this way plants detoxify Tl in the above-ground parts. Leaves were much more hairy in the Bolesław plants. This is an adaptation for growth in the extreme conditions of the zinc-lead waste heap with elevated Tl quantity.