The sequences of nearly 6,000 taxonomically described species and bacteria have been reclassified according to their 16S ribosomal DNA (rDNA) sequences. As a result, ribosomal RNA sequencing has become a powerful method for determining the phylogenetic position of unidentified strains. All together, the sequences of more than 60,000 16S rDNAs covering more than 97% of the sequences of officially described species has been accumulated. This data has revealed that many established species have nearly identical rRNA sequences. Thus, given sequencing errors and variations among the five to eight copies of the 16S rDNA operon on a chromosome from a single species, it is practically impossible to identify an isolated strain by sequencing if the strain less than 2% different than a closely related established species. The Ad Hoc Committee of the International Systematic Bacteriology recommended that, if the 16S rDNA sequence variation between a new strain and an established species was less than 3%, chromosomal DNA-DNA hybridization data is needed to describe the new species (18). Since the last century, members of the family Enterobacteriaceae have been classified according to their biochemical traits (3). For this reason, genetically identical organisms, such as four species of genus Shigella and Escherichia coli, had been misclassified as different species (26,27). Also, early in the 20th century, more than 2,000 Salmonella serovars had been classified as independent species, but they are now known to be a single species (33).Chromosomal DNA-DNA hybridization can solve the problems of phenotypic identification and 16S rDNA sequencing. A bacterial species is genetically defined as a group of strains that shares more than 70% Abstract: Quantitative DNA-DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3-labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA-DNA hybridization for measuring chromosome relatedness among bacterial species.