From the perspective of the biorefinery, the inherent heterogeneity of mixed-species feedstocks increases the apparent risk associated with their use due to the difficulty in predicting processing characteristics, potential yields and digestibility. These materials often contain species from different botanical classifications, which could affect pretreatment efficiency and Saccharification yields. For this study, we evaluated the impact of ammonia fiber expansion (AFEX™1) pretreatment conditions on hydrolysis sugar yields for five early successional old-field treatment replicates, each comprised of a different mixture of annual forb and grass species. Yields from these samples were also compared to a late successional old field sample and to corn stover that had been pretreated at the same conditions. Relatively high sugar yields were obtained for four of the five early successional feedstocks when pretreated at the same conditions: 2.0 g NH3:g DM; 0.5 g H2O:g DM; 100 °C; 30 min. It was found that glucan digestibility was strongly inversely correlated to the total lignin content; however this was not true of the xylan digestibility. The mixed-species feedstocks that had a higher grass content tended to also have a higher structural sugar content and were more digestible than forb-dominated feedstocks, thereby resulting in higher saccharification yields. Also, the grass-dominated mixed-species feedstocks were as digestible as corn stover (∼80% total sugar yields). However, due to its higher structural sugar content, corn stover had higher total glucose and xylose yields (580 g/kg biomass) compared to the mixed-species feedstocks, which ranged from 290 to 470 g/kg biomass.