The known manganese (Mn) hyperaccumulator Gossia bidwillii, and G. acmenoides unknown to hyperaccumulate Mn are tree species native to subtropical eastern Australia, where they co-occur on Mn-rich soils. Here, we investigate Mn accumulation and distribution in G. acmenoides collected from its natural habitat, and propagated G. bidwillii plants in a Mn dosing trial. Gossia bidwillii were subjected to different levels of Mn (250 µg g-1, 500 µg g-1, 1000 µg g-1) treatments whereas G. acmenoides were sampled from Mn rich soil. We used laboratory-based X-ray Florescence Microscopy (XFM) to elucidate in situ distribution patterns of Mn and other elements in hydrated G. acmenoides and G. bidwillii tissues. Data from G. acmenoides revealed that contrary to existing knowledge, it can be strongly Mn-hyperaccumulating, with foliar Mn concentrations of 39 000 µg g-1 and 24 000 µg g-1 in old and young leaves, respectively. In the Mn dosing trial, G. bidwillii accumulated 24 400 µg g-1 in old leaves and 17 100 µg g-1 in young leaves at the highest treatment level. The XFM data revealed clear interspecies differences in foliar Mn distribution patterns, with wild G. acmenoides leaves uniformly enriched throughout the laminae and petioles of both young and old leaves; while in G. bidwillii, the foliar Mn distribution was primarily concentrated at the apex and lamina. The approach employed of combining data from the field and controlled experiments was especially effective for comparing Mn accumulation in these two species and gaining added insight into the phenomenon of Mn hyperaccumulation.