The capability of alternating current (AC) dielectrophoresis (DEP) for on-chip capture and chaining of the three species representative of freshwater phytoplankton was evaluated. The effects of the AC field intensity, frequency and duration on the chaining efficiency and chain lengths of green alga Chlamydomonas reinhardtii, cyanobacterium Synechocystis sp. and diatom Cyclotella meneghiniana were characterized systematically. C. reinhardtii showed an increase of the chaining efficiency from 100 Hz to 500 kHz at all field intensities; C. meneghiniana presented a decrease of chaining efficiency from 100 Hz to 1 kHz followed by a significant increase from 1 kHz to 500 kHz, while Synechocystis sp. exhibited low chaining tendency at all frequencies and all field intensities. The experimentally-determined DEP response and cell alignment of each microorganism were in agreement with their effective polarizability. Mixtures of cells in equal proportion or 10-times excess of Synechocystis sp. showed important differences in terms of chaining efficiency and length of the chains compared with the results obtained when the cells were alone in suspension. While a constant degree of chaining was observed with the mixture of C. reinhardtii and C. meneghiniana, the presence of Synechocystis sp. in each mixture suppressed the formation of chains for the two other phytoplankton species. All of these results prove the potential of DEP to discriminate different phytoplankton species depending on their effective polarizability and to enable their manipulation, such as specific collection or separation in freshwater.