Ants possess many exocrine glands that produce a variety of compounds important for chemical communication. Fungus-growing ants, a tribe of over 230 species within the subfamily Myrmicinae, are unique among ants because they cultivate fungus gardens inside their nests as food. Here the chemistry of the exocrine glands of the two genera most closely related to the conspicuous leaf-cutting ants are examined. Based on a recent phylogeny of the fungus-growing ants, these genera comprise three clades that together link the more basal species to the most derived, leaf-cutting species. The leaf-cutting ants possess many derived characteristics such as extensive leaf-cutting behavior and massive colony sizes, effectively making them major herbivores in many Neotropical habitats. This is the first comparison of the chemistry of eight Trachymyrmex and one Sericomyrmex species in a phylogenetic context. Most of the compounds found in the Trachymyrmex species examined were terpenes. In one species, the major component was a,a-acariolide, the first example of this compound, which was only previously reported in mites, from an insect. Additionally, 3-octanol, 3-octanone, and 4-methyl-3-heptanone were detected, well-known mandibular gland compounds from a number of ant genera, together with high levels of undecane, likely from the Dufour’s gland, all generally thought to be used as alarm pheromones. Overall the combination of compounds discovered was unique for each species but biosynthetic similarities corroborate, at a very basic level, the phylogenetic relationships.