This study shows thallium (Tl) concentrations in Brassica juncea (Indian mustard) tissue are more than an order of magnitude higher (3830 μg/kg) than that of the substrate (100 μg/kg) and are strongly influenced by the underlying mineralogy; i.e., Tl bioaccessibility depends on the mineral structure: K-feldspar > Mn nodule > hendricksite mica. The majority of Tl for all substrates is contained in edible parts of the plant, i.e., leaves (41% of total Tl, on average) ≥ flower stems (34%) > seed pods (11%) ≈ stems (10%) > flowers (3%). We also show that Tl isotope fractionation induced by B. juncea is substantial, at nearly 10 εTl units, and generates systematic plant-specific patterns. Progressive plant growth strongly fractionates Tl isotopes, discriminating against Tl as the plant matures. Thus, Tl values are systematically higher in the early formed stem (εTl = +2.5) than in plant elements formed later (εTl = -2.5 to +0.1), which demonstrates the large degree of translocation and the associated effects during plant growth. This study establishes the potential of Tl isotopes as a new tool for understanding heavy metal (re)distribution during anthropogenic and geologic processes and the utility of such information in environmental and health-related planning and in phytomining or bioprospecting.