Pteris vittata (Chinese brake fern), the first reported arsenic (As) hyperaccumulating plant, can be potentially applied in the phytoremediation As-contaminated sites. Understanding the mechanisms of As tolerance and detoxification in this plant is critical to further enhance its capability of As hyperaccumulation. In this study, an unknown As species, other than arsenite (AsIII) or arsenate (AsV) was found in leaflets by using anion-exchange chromatography-hydride generation-atomic fluorescence spectroscopy and size-exclusion chromatography-atomic fluorescence spectrometry. The chromatographic behavior of this unknown As species and its stability suggest that it is likely an As complex. Although phytochelatin with two subunits (PC2) was the only major thiol in P. vittata under As exposure, this unknown As complex was unlikely to be an AsIII-PC2 complex by comparison of their chromatographic behaviors, stability at different pHs and charge states. The complex is sensitive to temperature and metal ions, but relatively insensitive to pH. In buffer solution of pH 5.9, it is present in a neutral form.