Aphids have developed symbiotic associations with different bacterial species, and some morphological and molecular analyses have provided evidence of the host relationship between the primary symbiotic bacteria (Buchnera aphidicola) and the aphid while the contrary with the secondary symbiotic bacteria. In this study, we investigated the phylogenetic relationships of the bacterial endosymbionts in the aphid Sitobion avenae (F.). We characterized all bacterial endosymbionts in 10 genetically defined S. avenae clones by denaturing gradient gel electrophoresis and, from these clones, sequenced the 16S rRNA genes of both the primary endosymbiont, B. aphidicola (for the first time), and the secondary endosymbionts, Regiella insecticola and Hamiltonella defensa (for the first time). The phylogenetic analysis indicated that Buchnera from Sitobion related to those in Macrosiphoni. The analysis of the secondary endosymbionts indicated that there is no host relationship between H. defensa and R. insecticola from Sitobion and those from other aphid species. In this study, therefore, we identified further evidence for the relationship between Buchnera and its host and reported a relationship within the secondary endosymbionts of S. avenae from the same country, even though there were no relationships between the secondary bacteria and their host. We also discussed the diversity within the symbiotic bacteria in S. avenae clones.