AbstractCytogenetic data on stingless bees outside the Neotropical region are still scarce, with chromosome numbers known for only a handful of species, without any further cytogenetic investigations. Our goal was to broaden the cytogenetic characterization of Austroplebeia australis from Australia to contribute to the cytogenetic knowledge of the Meliponini tribe. The following techniques were performed: Giemsa-staining, C-banding, DAPI/CMA3, and fluorescence in situ hybridization (FISH) with 18S rDNA, GA(15), GAG(10), CAA(10), CGG(10), and TTAGG(6) probes. Austroplebeia australis has 2n = 36 chromosomes and high heterochromatin content. DAPI+ coincided with euchromatic regions, whereas CMA3+ marked the heterochromatin. 18S rDNA clusters were identified in four chromosome pairs, and microsatellites were mainly present in the euchromatin. It has been hypothesized that 2n = 36 is the ancestral karyotype of the Meliponini tribe, and A. australis retains this plesiomorphic number. A large amount of heterochromatin is a common pattern observed in other stingless bees although it evolved independently in each genus. Interestingly, CMA3+ heterochromatin trait is shared with solitary bee species of the Euglossini and Emphorini tribes, whereas the Neotropical Meliponini species have heterochromatin DAPI+. The predominance of microsatellite sequences in euchromatin is a trend observed in Neotropical Meliponini, whereas TTAGG represents the telomeric sequence of bees and ants. This is the first study to investigate the cytogenetic patterns of a stingless bee outside the Neotropical region using different banding techniques. This body of work broadens Meliponini cytogenetic data and contributes to the identification of general evolutionary trends in this tribe.