Yerba mate is traditionally consumed in South America, and it has recently been highly publicized for its health benefits; however, consumers have felt differences in bitterness even in products of the same brand. The aim of this research was to compare the intensity of bitterness in native leaves and in cultivated leaves from 5- and 15-year-old trees, and determine the content of caffeine, reducing sugars and tannin in yerba mate leaves to explain their influence in bitterness. Bitterness was associated with age and cultivation of the plant, both of which can contribute to different blends of yerba mate. Sensory analysis revealed a significant difference between 5-year-old cultivated leaves and leaves from native trees and that bitterness decreases according to the age of the plant. Chemical analysis showed that caffeine and sugar content have no influence on its bitterness. However, tannin concentration is higher in younger plants, which are considered more bitter than older plants.