The aim of the research was to study and compare iodine concentration in natural waters originating from aquifers of different ages, primarily residents use for drinking purposes. The analysis was based on the original data on the samples collected during the fieldwork in the Bryansk region (2013–2017) and in the Oryol region (2016–2017). In addition to iodine concentration, the main geochemical parameters (salinity, etc.) were determined in the selected waters. The results showed a significant variation of iodine in waters from both regions (Bryansk region—from 0.7 to 41.2 µg/l; Oryol region—from 1.12 to 36.8 µg/l), the difference being apparently due to different ages and origins of the sampled aquifers (Quaternary, Upper Devonian and Cretaceous deposits). The overall low provision of surface water with iodine was found both in the Bryansk region (median for shallow wells—5.82 µg/l, median for surface water—6.76 µg/l) and in the Oryol region (median for shallow wells—2.96 µg/l, median for surface water—7.4 µg/l). The data obtained deserve attention during organization of monitoring and implementation of measures preventing thyroid diseases. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.