Aims: The aims of this study were to evaluate the host-specific distribution of Bacteroidales 16S rRNA gene sequences from human- and animal-related effluents and faeces, and to define a ruminant-specific marker. Methods and Results: Bacteroidales 16S rRNA gene clone libraries were constructed from samples of effluent (sewage, bovine manure and pig slurry) and faeces (human, bovine, pig and wild bird), using PCR primers targeting order Bacteroidales. The phylogenetic analysis revealed six main distinct human-, bovine-, pig- and wild bird-specific clusters. From the bovine-specific cluster II, we designed a ruminant-specific marker, Rum-2-Bac, and this showed 97% sensitivity (n = 30) and 100% specificity (n = 40) when tested by TaqMan (R) real-time PCR. Average concentrations of this marker in bovine and sheep faeces and in bovine manure were 8 center dot 2 +/- 0 center dot 5, 8 center dot 4 +/- 1 center dot 3 and 7 +/- 0 center dot 5 log(10) copies per gram, respectively. It was also quantified in samples of runoff water impacted by bovine manure, with average concentrations of 5 center dot 1 +/- 0 center dot 3 log(10) copies per millilitre water. Conclusions: Our results confirmed that some members of Bacteroidales isolated from effluents and faeces had host-specific distributions. Identification of a bovine-specific cluster made it possible to design a reliable ruminant-specific marker. Significance and Impact of the Study: The host-specific distribution of Bacteroidales sequences from effluents mirrored the host-specific distribution of sequences observed in individual faeces. This efficient new ruminant-specific Bacteroidales 16S rRNA marker represents a useful addition to the microbial source tracking toolbox.