The biology and ecology of four medicinal species of Gentianella (Gentianaceae) were described, with emphasis on aspects related to production, including the analysis of total flavonoids. All the evaluated species were small grasses. Gentianella graminea and G. crassicaulis are perennial and G. dianthoides and G. bicolor are annual. While G. dianthoides produces abundant seeds, the others have little to moderate seed production, all with low germination capacity. Gentianella crassicaulis produces up to 90 g of dry matter/plant, while the rest produces less than 10 g of dry matter/plant. Gentianella graminea presented significantly higher levels of total flavonoids in leaves and stems compared to G. dianthoides and G. crassicaulis (p < 0,05). Three species (G. graminea, G. crassicaulis and G. dianthoides) occur in the Jalca region (2700 to 2850 m a.s.l.), along with other 30 species (of which 16 medicinal); while G. bicolor inhabits the Quechua region (2600 a 2750 m a.s.l.), along with 17 species (9 medicinal). The four species had scarce populations (≤ 2,5 plants/m2) and their natural regeneration was low (1-4 plants/m2). Gentianella dianthoides and G. crassicaulis are in a vulnerable state of conservation, so it is recommended to suspend their collection.