The Bacterial and Archaeal communities in a 1.14 m3 ambient temperature anaerobic digester treating dairy cow manure were investigated using terminal restriction fragment length polymorphisms (T-RFLP) and direct sequencing of the cloned polymerase chain reaction (PCR) products. Results indicate shifts in the structure of the both the Archaeal and Bacterial communities coincided with digester re-inoculation as well as temperature and loading rate changes. Following re-inoculation of the sour digester, the predominant Archaea shifted from Methanobrevibacter to Methanosarcina, which was the most abundant Archaea in the inoculum. Methonosarcina was replaced by Methanosaeta after the resumption of digester loading in the summer of 2010. Methanosaeta began to decline in abundance as the digester temperature cooled in the fall of 2010 while Methanobrevibacter increased in abundance. The microbial community rate of change was variable during the study period, with the most rapid changes occurring after re-inoculation.