Agave plants, which grow well in warm semi-arid lands around the world, are well known for production of tequila and other alcoholic beverage production in Mexico. While residues are being tested for biofuel production, no Agave species able to survive freezing conditions, like common to the US southwest, have been used for biofuel production likely because hydrolysates from most Agave species are lethal to yeast. Through testing, we have developed a two-step process that eliminated this toxicity allowing production of ethanol, as well as n-butanol from multiple Agave species, some cold tolerant to at least -18ºC. This process hydrolyzed and removed the liberated Agave sugars, especially acid sensitive fructose, prior to acidic pretreatment, hydrolysis and fermentation of the solids, thus maximizing the biofuel yields from the whole Agave plant. Analysis of different steps of the process determined the distribution and level of six biomass sugars for ten Agave species, with fructose up to about 30% of the carbohydrates. Additionally, yeast readily produced ethanol from ten whole Agaves hydrolysates produced using a special blend of biomass degrading enzymes that included inulinase, which removed the inhibition. For example, US-grown Agave neomexicana produced 119 ± 11 mg ethanol/g biomass and with Clostridium beijerinckii 144 ± 4 mg butanol plus other fermentation products/g biomass. Using this new two-step process, there is high potential to utilize numerous high yielding Agave species growing in both tropical and temperate semi-arid regions around the world as sources of high levels of fermentation biofuels and biochemicals.