The plant-pathogenic bacterium Xylella fastidiosa which causes significant diseases to various plant species worldwide, is exclusively transmitted by xylem sap-feeding insects. Given the fact that X. fastidiosa poses a serious potential threat for olive cultivation in Greece, the main aim of this study was to investigate the genetic variation of Greek populations of three spittlebug species (Philaenus spumarius, P. signatus and Neophilaenus campestris), by examining the molecular markers Cytochrome Oxidase I, cytochrome b and Internal Transcribed Spacer. Moreover, the infection status of the secondary endosymbionts Wolbachia, Arsenophonus, Hamiltonella, Cardinium and Rickettsia, among these populations, was determined. According to the results, the ITS2 region was the less polymorphic, while the analyzed fragments of COI and cytb genes, displayed high genetic diversity. The phylogenetic analysis placed the Greek populations of P. spumarius into the previously obtained Southwest clade in Europe. The analysis of the bacterial diversity revealed a diverse infection status. Rickettsia was the most predominant endosymbiont while Cardinium was totally absent from all examined populations. Philaenus spumarius harbored Rickettsia, Arsenophonus, Hamiltonella and Wolbachia, N. campestris carried Rickettsia, Hamiltonella and Wolbachia while P. signatus was infected only by Rickettsia. The results of this study will provide an important knowledge resource for understanding the population dynamics of vectors of X. fastidiosa with a view to formulate effective management strategies towards the bacterium.