While studies have shown that anaerobic co-digestion of chicken manure (CM) and corn stover (CS) is an efficient method to treat these agricultural wastes, the microbial ecology of these systems and optimal parameters for the digestion process are yet to be determined. In this study, the effects of different initial substrate concentrations and CS : CM mixture ratios on co-digestion and microbial community structure were evaluated. Results demonstrated that both the highest cumulative methane yields and methane production rates were obtained from reactors with a CS : CM ratio of 1 : 1 during hemi-solid-state anaerobic digestion (HSS-AD). Cumulative methane yields and methane production rates were 24.8% and 42% lower in solid-state anaerobic digestion (SS-AD) reactors using the same CS : CM ratios. Analysis of microbial community structures revealed that cellulolytic bacteria and a diversity of syntrophic microorganisms capable of direct interspecies electron transfer (DIET) and hydrogen interspecies transfer (HIT) were enriched in the best-performing reactors. Methanosarcina species also dominated during HSS-AD, and their presence was positively correlated with methane production in the reactors.