A variety of plant associated beneficial bacteria (PABB) occur in rhizosphere of many plants. This study developed an oligonucleotide microarray for detection and identification of these PABBs and evaluated the oligonucleotide microarray with 43 pure cultures of PABB which were well-identified by sequencing the initial 600-800bp of the 16S rRNA gene. Ninety six species- or genus-specific oligonucleotide probes targeting 16S rRNA gene of PABB and three control probes were spotted onto silylated glass slides. 16S rDNA sequences of the reference strains were PCR amplified by a pair of bacterial universal primers, and the amplicons were labeled with Cy5-dUTP and hybridized with the oligonucleotide microarray. After hybridization, the microarray was scanned to detect the green Cy5-dUTP signals and generated a series of typical species- or genus-specific hybridization profiles. The microarray was able to differentiate closely related strains, indicating that the oligonucleotide microarray method developed in this study is a useful tool for the detection of pure cultures of PABB strains.