Baker's yeast (Saccharomyces cerevisiae) was used as substrate for the selective retention of Cr(III) in the presence of Cr(VI) from aqueous solutions. Experiments were performed in batch as a suitable and simple method to obtain information of chromium uptake. The effect of chemical and physical variables affecting the degree of uptake of chromium was tested in order to select the optimal analytical conditions for the selective Cr retention by Saccharomyces cerevisiae. The parameters studied were: reaction media and pH, amount of biomass, temperature and contact time. The influence of some concomitant ions up to 50 mg L^-1 was also tested. During all the steps of the optimization process, Cr(VI) remained in solution while Cr(III) was accumulated by the yeast cells. Quantitative determinations of Cr species in the biomass and supernatant solution were carried out by means of flame atomic absorption spectrometry (FAAS). A preconcentration factor of 15 was achieved for Cr(III) when 75 mL of water were processed. The detection limits for Cr(III) and Cr(VI) were 0.2 and 3.0 ng mL^-1 respectively. Compared with other methods, this approach allows the determination of Cr(III) and Cr(VI) in different phases. A morphological study and microscopical characterization of the yeast cell wall was carried out by Scanning Electron Microscopy (SEM) complemented with energy dispersive X-ray analysis (EDAX). The speciation analysis of inorganic Cr in different kinds of spiked natural waters was performed following the proposed method. Recoveries in each phase were in all cases between 89 and 108%.