The establishment of a sensitive and specific method for the detection of reduced phosphorus (P) is crucial for understanding P cycle. This paper presents the quantitative evidence of phosphite (P, +3) from the freshwater matrix correspondent to the typically eutrophic Lake Taihu in China. By ion chromatography coupled with gradient elution procedure, efficient separation of micromolar levels of phosphite is possible in the presence of millimolar levels of interfering ions, such as chloride, sulfate, and hydrogen carbonate in freshwater lakes. Optimal suppressed ion chromatography conditions include the use of 500 μL injection volumes and an AS11 HC analytical column heated to 30 °C. The method detection limit of 0.002 μM for phosphite was successfully applied for phosphite determination in natural water samples with recoveries ranging from 90.7 ± 3.2% to 108 ± 1.5%. Phosphite in the freshwater matrix was also verified using a two-dimensional capillary ion chromatography and ion chromatography coupled with mass spectrometry. Results confirmed the presence of phosphite in Lake Taihu ranging from 0.01 ± 0.01 to 0.17 ± 0.01 μM, which correlated to 1-10% of the phosphate. Phosphite is an important component of P and may influence biogeochemical P cycle in lakes.