The arsenic (As) hyperaccumulator Pteris vittata has super As accumulation ability and huge biomass, thus being ideal plant material for the phytoremediation of As-contaminated soil. Phytoextraction technology based on As hyperaccumulator P. vittata has been applied to more than 20 soil remediation projects in China. So far, the reported P. vittata populations all showed strong accumulation ability of As, and this hyperaccumulation ability was able to be stably inherited from the progenitor. How can this fern pass As hyperaccumulation ability on to the next generation through spores (the germ cell of fern plants) with the size of several micrometers? Compared to the traditional chemical analysis methods, Synchrotron Radiation X-ray Fluorescence Microprobe Analysis and X-Ray Absorption Spectroscopy show high sensitivity and are easy to operate, recently widely used to study hyperaccumulating mechanisms. This study investigated the micro-distribution pattern of As in spores using Synchrotron Radiation X-ray Fluorescence Microprobe Analysis micro-speciation of As in spores using Synchrotron Radiation X-ray Absorption Spectroscopy. The distribution of As was compared to that of potassium, calcium, iron, sulfur, copper and zinc. It has been found that the distribution of As, sulfur, and calcium was similar. It was indicating the role of their interaction in the stable inheritance of As hyperaccumulation. The micro speciation analysis indicated that the main species of As in spores and sporangium was arsenite (AsⅢ). Considering that AsⅢ has higher mobility and toxicity than arsenate in most organisms, the results indicated high tolerance of P. vittata to As throughout its life cycle. Using X-ray Fluorescence Microprobe Analysis and X-ray Absorption Spectroscopy with high resolution, results provide basic information for understanding the genetic characteristics of arsenic hyperaccumulation ability of P. vittata.