In recent years, ambrosia beetles, Euwallacea interjectus, have emerged as important pests of exotic Populus in poplar cultivations from Jiangsu, China. However, Fusarium mutualists of E. interjectus within the Ambrosia Fusarium Clade (AFC) remain unclear. In the investigation of Fusarium mutualists, 36 isolates were obtained from five geographically separated sites in Jiangsu Province and Jiangxi Province. Three novel species of AFC were identified based on morphological and multi-gene phylogenetic analysis; three novel species of AFC were identified. These new species are described here as Fusarium gannanense (AF-25), Fusarium populicola (AF-26), and Fusarium tumidispermus (AF-27). Fusarium populicola may be a pathogen of poplar and exhibited common to poplar cultivations in Jiangsu Province. The other two species were recovered from three locations in Jiangxi Province. The distribution of mutualists of E. interjectus within AFC is associated with the geographical distance among the samples with samples from the same region sharing the same mutualist. The present study augments our knowledge on the mutualists of Euwallacea within AFC in the native geographic range.