Aims: The aim of the study was to develop a microarray dedicated to comprehensive analysis of the diverse rumen bacteria. Methods and Results: All the 16S rRNA gene (rrs) sequences of rumen origin were retrieved from the RDP database, and operational taxonomic units (OTUs) were calculated at 97% sequence similarity. A total of 1666 OTU-specific probes were designed and synthesized on microarray slides (referred to as RumenBactArray) in a 6 x 5 k format with each probe being represented in triplicate. The specificity, sensitivity and linear range of detection were determined using pools of rrs clones of known sequences. The RumenBactArray detected as few as approx. 10(6) copies of a target and had a linear detection range of > 4 orders of magnitude. The utility of the RumenBactArray was tested using fractionated rumen samples obtained from sheep fed two different diets. More than 300 different OTUs were detected across the four fractionated samples, and differences in bacterial communities were found between the two diets. Conclusions: This is the first phylochip dedicated to analysis of ruminal bacteria, and it enables comprehensive and semiquantitative analysis of ruminal bacteria. Significance and Impact of the Study: RumenBactArray can be a robust tool to comparatively analyse ruminal bacteria needed in nutritional studies of ruminant animals.