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Amomum xanthioides Wall. is a valuable medicinal plant, used in several medical prescriptions. Study on the culture of callus biomass of this species has important implications, contributing to the supply of cell biomass for the study of extracting the valuable natural bioactive compounds. This is the first report on the in vitro callus culture of Amomum xanthioides Wall. In this study, callus was induced from explant types of natural plant (nodal stem and seeds) and in vitroshoots (stem node, leaf sheath and shoots tip) cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of dichorophenoxyacetic acid (2,4-D), benzylaminopurine (BAP) and malt extract (ME). The result indicated that in vitro shoot tip is the most suitable tissue for callus formation than other explant types. The highest callus biomass was observed on callus culture medium supplemented with 1.5 mg/l 2,4-D and 0.5 mg/l BAP with the presence of 500 mg/l malt extracts (ME). In this study, the rapid protocol for Amomum xanthioides callus production was established, that could help in the study of large-scale production of bioactive compounds for valuable pharmaceutical applications from this rare species.