Chemical fractionation is generally used to assess the mobility and bioavailability of heavy metals present in solid environmental samples. Although fractionation results are determined by sample pretreatment methods, authors of publications usually disregard this issue and study exclusively the distribution of metals among biogeochemical phases. The aim of this work was to assess the effect of preparation of samples on results of chemical fractionation of chromium deposited in river sediments contaminated with tannery wastes. Attention was focused on the evaluation of the effect of drying and grinding of sediments. Fractionation was carried out according to the procedure of Tessier which makes it possible to divide total chromium content into five fractions of different mobility and bioavailability (ion-exchange, carbonate, oxide, organic and residual fractions). It has been found that sediment grinding is not an appropriate method of sample pretreatment, because it significantly changes fractionation results and thus it can lead to an incorrect assessment of mobility and bioavailability of chromium deposited in river sediments. Drying of the sediments at a range of 20–105 °C does not change the original distribution of chromium among biogeochemical phases.