Ascomycete fungi in the nests of ants inhabiting plants (= myrmecophytes) are very often cultivated by the ants in small patches and used as food source. Where these fungi come from is not known yet. Two scenarios of fungus recruitment are possible: (1) random infection through spores or hyphal fragments from the environment, or (2) transmission from mother to daughter colonies by the foundress queen. It is also not known at which stage of the colony life cycle fungiculture is initiated, and whether the- symbiont fungi serve as food for the ant queen. To clarify these questions, we investigated four Azteca ant species inhabiting three different Cecropia species (C. insignis, C. obtusifolia, and C. peltata). We analysed an rRNA gene fragment from 52 fungal patches produced by founding queens and compared them with those from established Azteca colonies (n = 54). The infrabuccal pockets of winged queens were dissected to investigate whether young queens carry fungi from their mother colony. Additionally, 15N labelling experiments were done to verify whether the queen feeds on the patches until she is nourished by her first worker offspring. We infer from the results that the fungi cultivated in hollow plant structures are transferred from the parental colony of the young queen. First, fungal genotypes/OTU diversity was not significantly different between foundress queen patches and established colonies, and second, hyphal parts were discovered in the infrabuccal pockets of female alates. We could show that fungiculture already starts before queens lay their eggs, and that the queens do not feed on fungal patch material but feed it to the larvae. Our findings suggest that fungiculture may be crucial for successful colony founding of arboreal ants in the tropics.