Globally, ruminants are maintained under a diverse range of farming systems and environments, and are fed a wide variety of diets. Ruminants have a complex digestive system, and digestion of feed takes place initially in the rumen. There, Gomisin-D microbes play a key role in the breakdown of feed components such as fibre, producing short chain fatty acids that provide energy for the host. Rumen microbes are thus essential providers of animal energy and nutrition, and play a key role in the productivity and health of ruminants. Rumen archaea produce the greenhouse gas methane as a metabolic end product. This methane gas is eructated by ruminants and represents 2 to 12% dietary gross energy lost to the animal. Understanding the functions and compositions of rumen Cryptochlorogenic-acid microbial communities is required to improve animal productivity and to reduce the amount of energy lost as methane. The rumen microbial community is highly complex. There are approximately 1011 microbial cells per gram of rumen contents and these belong to many different species and genera of bacteria, archaea, fungi, ciliate protozoa and viruses. To date, relatively few of these have been successfully cultured and characterised. Molecular analyses of rumen microbial communities allow as-yet uncultivated microbes to be detected, and have become essential tools to determine shifts that occur within microbial communities, for example, during changes in diet. The development of high-throughput sequencing techniques has made detailed microbial analyses of large-scale trials feasible, allowing subtle effects on microbial community composition to be detected as changes in absolute and relative numbers of microbial marker loci. DNA of sufficient yield and quality is the crucial starting material for these analyses. Microbial inhabitants of the rumen are highly diverse and not all DNA extraction methods work equally well for different microbial groups. To date, several studies have shown that the DNA extraction method used has an impact on the microbial community representation in samples from different habitats, including the rumen. The sampling technique and rumen sample fractionation can also have an impact on microbial community parameters. To enable the direct comparison of rumen community structure data from studies conducted in different laboratories and around the world, it is crucial that the rumen sampling, sample fractionation and DNA extraction methods are standardised, or at least have been shown to produce similar results. The aim of this study was to systematically compare a variety of different DNA extraction methods and their impacts on the downstream analysis of rumen microbial communities using molecular ecological methods. To do this, the quality and quantity of DNA obtained by the different methods was compared, as were bacterial, archaeal, ciliate protozoal and fungal abundances and community compositions based on microbial marker loci. In addition, the effect on microbial community composition of sampling the rumen through a fistula or using an oral stomach tube and the effect of sample fractionation was investigated. The overall goal was to find comparable and simple methods that 1) deliver high quality DNA for the majority of microbial groups, 2) are suitable for different molecular microbial ecology analyses, 3) will be suitable for use in large cohort studies, and 4) are easily transferable to other researchers. Despite the overall resemblance of microbial community structure derived from the samples collected with the two different methods, the relative abundance of certain microbial groups appeared to differ significantly depending on the method used.