Andean Lupin (Lupinus mutabilis S.) or tarwi, a legume cultivated in the Andean region of South America, is considered a protein source. In this work, flours were prepared from different Andean Lupin genotypes, namely Cholo Fuerte (CHF), Común (CM), or Altagracia (ALT), grown at 2700 (1) or 3300 (2) meters above sea level. The effect of genotype and growth altitude on the chemical composition, color, phytochemical (alkaloids, free phenolics, fatty acids, and sterols) content, antioxidant activity, and microbiological quality of the flours was evaluated. CM-1 flour had higher protein, polyunsaturated fatty acid (PUFA), and Δ5-avenasterol content. CHF-1 flour had higher crude fiber, Δ7-campesterol, clerosterol, and saturated fatty acid (SFA) content and higher antioxidant activity. ALT-2 flour had the highest total sterol content, particularly stigmasterol and β-sitosterol. None of the flours contained alkaloids or microorganisms, confirming that the preparation process was efficient. Andean Lupin cultivated at high and low altitude provides flour with high phytosterol and PUFA content, respectively. CM-1 flour has potential use in the food and nutraceutical industry due to its higher protein, essential fatty acid or PUFA, and total sterol content as well as low SFA content.