Large variations in pre-nuclear ¹²⁹I/¹²⁷I ratios in terrestrial environments have been observed, but few investigations have been carried out on how the isotopic composition varies and how it is affected by the terrestrial environment. In this work, lake and river sediments were studied for the first time with the aim of exploring the possible connections between the natural iodine isotopic composition and its sources. The observed higher natural ¹²⁹I/¹²⁷I ratios compared to those in the marine system suggested that the isotopes did not reach a steady state. Decreasing ¹²⁹I/¹²⁷I ratios with increasing ¹²⁷I contents indicated perturbation of old iodine, and a narrower range of initial equilibrium ratios was suggested based on pre-nuclear ratios derived in the studied sediments.