A hyphenated instrumental method high-performance liquid chromatography-inductively coupled plasma-optical emission spectrometry (HPLC-ICP-OES) was used for the separation and determination of Cr(III) and Cr(VI). After the chromatographic separation of chromium species by anion exchange column, their spectrophotometric detection was carried out by ICP-OES system. Important instrumental and chromatographic parameters were investigated via univariate optimization approach to obtain high signal to noise ratio and good resolution for chromium species. Under the optimum HPLC-ICP-OES conditions, limit of detection (LOD) values for Cr(III) and Cr(VI) were found to be 0.27 and 0.05 mg/kg, respectively. In addition, accuracy and applicability of developed method were checked by recovery experiments performed with the spiked soil, grass, and water samples. High percent recovery results (88-104%) were achieved by utilizing matrix matching calibration strategy for the selected samples. The results showed that this method was accurate and applicable to soil, grass, and water samples.