Agave durangensis propagates basically by seeds. This species is economically important because it supports a mescal industry in Durango, Mexico. At present, its exploitation is overall by collecting plants from the wild populations. In order to reduce the negative impact of the collection in the natural populations of A. durangensis and to improve the mescal industry, it is necessary to establish plantations with selected seeds for a high vigor. In this paper, the variation in morphological features, physiological behavior of germination, and biochemical indicators of seed vigor among three natural populations of A. durangensis was assessed. Variation was found in the seed weight (0.68 to 1.15 mg/seed), seed dimensions (3.51 × 5.29 to 4.62 × 5.92 mm), germinability reduction at 15˚C related to 25˚C (4% to 51%), germination rate at 25˚C (44.0 to 48.5 seeds/day) and at 15˚C (5.17 to 6.77 seeds/day), development reduction at 15˚C related to 25˚C (86.66% to 91.99%), levels of seed accumulated phenols (71 to 85 µg/seed), antioxidant potential (42% to 50% reduction of DPPH * ), seed alcohol dehydrogenase activity (ADH) (180 to 1100 µmol NAD + /mg protein/min), highest ADH activity after imbibitions at 25˚C (310.24 to 520.2 µmol NAD + /mg protein/min), and highest ADH activity after imbibitions at 15˚C (170.74 to 440.71 µmol NAD + /mg protein/min). The variation in the seed vigor was revealed by a principal component analysis (PCA) based on all the parameters evaluated. PCA clearly discriminated among the three populations.