DNA molecular hybridization of chromosomal DNA among 15 strains of Brachybacterium were carried out by the method in which DNA labeled with nonradioactive photobiotin was binded to heat denatured single-stranded DNA in microdilution plates. Homology values among bacteria were obtained by measuring fluorescence intensity in the microdilution wells.The strains which is difficult to further classify at the species level with morphological, physiological and biochemical characteristics were determined their appropriate taxonomic positions on the basis of the genetic relatedness among microorganisms. This method is a rapid and accurate hybridization technique and plays diagnostic role on the taxonomic study of Brachybacterium.