The ecdysteroid 20-hydroxyecdysone (20E) is a steroid hormone found in insects and few plants. To analyze its temporal and spatial distribution in Pfaffia glomerata (Amaranthaceae), we selected among 71 accessions, the accession 13 which showed the highest root 20E total quantity (1.48 g/plant root). The 20E was constantly detected in flowers, leaves, stems, and roots, but its percentage was variable throughout its development. The highest 20E percentage was found in flowers (0.82%), roots (0.66%), leaves (0.60%), and stems (0.24%). While stems showed the least variable 20E percentage (0.13% - 0.24%), followed by roots (0.42% - 0.66%), and flowers (0.47% - 0.82%). Leaves showed the greatest 20E percentage variation (0.21% - 0.60%). The stem and leaves 20E total amount remained constant, showing a little variation while root 20E total amount increased over time, suggesting that 20E may be accumulated in roots. These findings suggest that 20E has tissue specific functions in plant and may have ecological significance since uncoordinated pulses of 20E are often lethal to insects.