The definition and delineation of microbial species are of great importance and challenge due to the extent of evolution and diversity. Whole-genome DNA–DNA hybridization is the cornerstone for defining procaryotic species relatedness, but obtaining pairwise DNA–DNA reassociation values for a comprehensive phylogenetic analysis of procaryotes is tedious and time consuming. A previously described microarray format containing whole-genomic DNA (the community genome array or CGA) was rigorously evaluated as a high-throughput alternative to the traditional DNA–DNA reassociation approach for delineating procaryotic species relationships. DNA similarities for multiple bacterial strains obtained with the CGA-based hybridization were comparable to those obtained with various traditional whole-genome hybridization methods (r=0.87, P<0.01). Significant linear relationships were also observed between the CGA-based genome similarities and those derived from small subunit (SSU) rRNA gene sequences (r=0.79, P<0.0001), gyrB sequences (r=0.95, P<0.0001) or REP- and BOX-PCR fingerprinting profiles (r=0.82, P<0.0001). The CGA hybridization-revealed species relationships in several representative genera, including Pseudomonas, Azoarcus and Shewanella, were largely congruent with previous classifications based on various conventional whole-genome DNA–DNA reassociation, SSU rRNA and/or gyrB analyses. These results suggest that CGA-based DNA–DNA hybridization could serve as a powerful, high-throughput format for determining species relatedness among microorganisms.The ISME Journal (2008) 2, 642–655; doi:10.1038/ismej.2008.23; published online 28 February 2008 [ABSTRACT FROM AUTHOR]