Tropical arboreal ants mainly feed as ‘secondary herbivores’, relying mostly on nitrogen-poor homopteran exudates as food. It has been speculated that this nitrogen-limitation of their diet may be overcome by nutritional upgrading with the help of symbiotic bacteria. We examined the bacterial diversity associated with several representatives of three species groups of the arboreal ant genus Tetraponera based on genes encoding 16S rRNA, citrate synthase and a structural protein of the dinitrogenase complex (nifH). The bacterial microflora showed group-specificity, suggesting long-term association between ants and bacteria. In all specimens of four species of the nigra group, we detected bacteria closely related to Bartonella (order Rhizobiales). Ants of the allaborans group harboured bacteria belonging to the enterobacteria. In Tetraponera pilosa of the third species group, we found the enterobacterium Pantoea agglomerans. In spite of the different phylogenetic affiliation of the bacteria identified in the three species groups, the presence of nifH in most species suggests a role in nitrogen metabolism of the bacterial microbiota. We also detected a high infection rate with the intracellular bacterium Wolbachia in all Tetraponera species groups. Besides the widespread bacteria found in Tetraponera, we discovered a diverse group of bacteria represented by single sequences. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 90, 399–412.