The excessive arsenic content in wild Cordyceps sinensis has caused great concerns on human health. The toxicity of arsenic depends on its concentration, chemical form, and valence. The source studies of arsenic in C. sinensis are essential for safety evolution and quality control. We used ICP-MS and HPLC-ICP-MS methods to determine the total arsenic amount and the arsenic speciation. Synchrotron-based XANES and micro-XRF imaging techniques were used to characterize arsenic valence and distribution. The total arsenic amount range in wild C. sinensis samples was 5.77–13.20 μg/g with an average of 8.85 ± 2.5 μg/g. As(III) and As(V) were the main species in wild C. sinensis samples. The iAs only accounts for 4.47–11.42% of the extracted arsenic. Trivalent and pentavalent forms were the dominant chemical forms of arsenic. Besides, we found that arsenic was accumulated at the digestive tract of the host larva.