In this study, the effect of total solid content (TS) on thermophilic hydrogen production from wheat straw was investigated. Six TS contents ranging from wet to dry conditions (10-34%TS) were tested in batch tests. A decrease of H₂ yields was observed and three statistical groups were distinguished according to the TS content: wet conditions (10% and 14%TS) with 15.3 ± 1.6 NmlH₂ gTS(-1), intermediate conditions (19%TS) with 6.4 ± 1.0 NmlH₂ gTS(-1) and dry conditions (25-34%TS) with 3.4 ± 0.8 NmlH₂ gTS(-1). Such a decrease in biohydrogen yields was related to a metabolic shift with an accumulation of lactic acid under dry conditions. Concomitantly, a microbial population shift was observed with a dominance of species related to the class Clostridia under wet conditions, and a co-dominance of members of Bacilli, Clostridia classes and Bacteroidetes phylum under dry conditions.