Xylella fastidiosa causes Pierce's disease (PD) and is transmitted by xylem-sap-feeding insects. While X. fastidiosa- infected grapevines have been detected, the transmission vectors reported have never been recorded in Taiwan. Previous studies have suggested that Kolla paulula (Walker) and Bothrogonia ferruginea (F.) are candidate vectors in Taiwan. Here, we explored the life history of these two leafhoppers, evaluated the transmission efficiency of X. fastidiosa by the vectors, and investigated the genetic identity of three collected X. fastidiosa strains, namely, GMb, BQa, and BQ7f from the grapevine cultivars Golden Muscat (GM) and Black Queen (BQ), and one previously extracted strain GV148 from Kyoho (GV) showing PD symptoms in local vineyards. The results showed that all four strains were 100% identical to X. fastidiosa isolate Temecula1 from a naturally infected grapevine in the United States based on sequence analyses of 16S rRNA and 16S-23S ITS. The acquisition rates by K. paulula and B. ferruginea from the symptomatic cultivar Golden Muscat were 83.3 and 70.0% per individual, and the transmission rates to healthy grapevines were 13.3 and 6.7%, respectively. The acquisition rates by the groups of three K. paulula from the symptomatic cultivars Golden Muscat and Black Queen were 54.7 and 49.6%, respectively. Additionally, the transmission rates by K. paulula from and to each of these two grapevine cultivars were not significantly different. In view of their acquisition from infected grapevines and the effective transmission of X. fastidiosa to healthy grapevines, these two sharpshooter species are vectors of X. fastidiosa in Taiwan.