Ammonia pretreatment greatly improves enzymatic hydrolysis of grass biomass, but is reported to be ineffective for hardwood biomass. Here, we examined the effectiveness of ammonia pretreatment of biomass from six hardwood species with different contents of xylan and lignin. Ammonia pretreatment greatly improved enzymatic hydrolysis of polysaccharides in birch and willow, but was less effective for acacia, eucalyptus, and poplar. The effectiveness of ammonia pretreatment increased with xylan content but decreased with lignin content of the hardwood species. By adding a recombinant xylanase to the commercial enzyme digestion cocktail, the yield of enzymatic hydrolysis of ammonia-pretreated birch biomass was improved to a similar level to that obtained with grass biomass. Our results indicate that enzymatic hydrolysis of biomass from hardwood species having a relatively high xylan/lignin ratio can be achieved with a xylanase-enriched enzyme cocktail after ammonia pretreatment.