Selenium is an important element in environmental and living organisms that is being essential in very narrow concentration range, while deficiency or toxicity occurs outside this range. However, its toxicity depends not only on its dose but also on its chemical form. In environmental samples, selenium can exist in inorganic forms (as elemental selenium, metal selenides, selenite, or selenate anions) and as organic species with direct C-Se bonds (methylated compounds, selenoaminoacids, and selenoproteins). Thus, the development of reliable techniques to study the speciation of selenium in environmental samples is necessary. The main purpose of this chapter is to provide an update on the recent literature concerning the strategies for selenium speciation in environmental samples. Liquid chromatography coupled with sensitive detector is a commonly used technique for selenium separation. Gas chromatography can also be applied for such purpose; however derivatization step is usually required before analysis. Direct determination of selenium species at the concentration levels present in natural samples is very often difficult or even impossible. For this, several preconcentration/separation procedures for selenium have been proposed, including coprecipitation, extraction into an organic solvent, or application of solid sorbents.