Technical Abstract: Spiroplasmas are bacteria in the Class Mollicutes that are frequently associated with insects and/or plants. Here, we identified apparent commensal spiroplasmas in the midgut and hindgut of five leafhopper species from laboratory-reared colonies. Those found in Dalbulus elimatus, Endria inimica and Macrosteles quadrilineatus were long and tubular shaped, whereas those in Dalbulus maidis and Graminella nigrifrons were shorter and mostly rod-shaped in their host organisms. These spiroplasmas were found in great numbers in the gut lumen frequently associated with the gut microvilli, but unlike the plant-pathogenic mollicutes, they did not seem to invade the gut epithelium or other tissues in any of these five leafhopper species. These spiroplasmas were more commonly found in males than in females and in crowded than in singly reared leafhoppers. The spiroplasmas were cultured and triply cloned for further taxonomic characterization. Sequence analyses of 16S rDNA genes and subsequent phylogenetic analyses revealed that all five Spiroplasma strains clustered with S. floricola. Serological analyses using the deformation test with antisera from spiroplasmas in the same phylogenetic cluster also indicated a close association with S. floricola. We speculate that these gut spiroplasmas are horizontally transmitted between leafhoppers during feeding, probing or grooming behavior.