Five strains, JPY461(T), JPY359, JPY389, DPU-3 and STM4206 were isolated from nitrogen-fixing nodules on the roots of Mimosa spp. and their taxonomic positions were investigated using a polyphasic approach. All five strains grew at 15-40 degrees C (optimum, 30-37 degrees C), at pH 4.0-8.0 (optimum, pH 6.0-7.0) and with 0-1% (w/v) NaCl [optimum, 0% (w/v)]. On the basis of 16S rRNA gene sequence analysis, a representative strain (JPY461(T)) showed 97.2% sequence similarity to the closest related species Burkholderia acidipaludis SA33(T), a similarity of 97.2% to Burkholderia terrae KMY02(T), 97.1% to Burkholderia phymatum STM815(T) and 97.1% to Burkholderia hospita LMG 20598(T). The predominant fatty acids of the five novel strains were summed feature 2 (comprising C-16:1 iso I and/or C-14:0 3-OH), summed feature 3 (comprising C-16:1 omega 7c and/or C-16:1 omega 6c), C-16:0, C-16:0 3-OH, C-17:0 cyclo, C-18:1 omega 7c and C-19:0 cyclo omega 8c. The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 63.0-65.0 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, an unidentified aminolipid and several unidentified phospholipids. The DNA-DNA relatedness of the novel strain with respect to recognized species of the genus Burkholderia was less than 54%. On the basis of 16S rRNA and recA gene sequence similarities, chemotaxonomic and phenotypic data, the five strains represent a novel species in the genus Burkholderia, for which the name Burkholderia diazotrophica sp. nov. is proposed with the type strain, JPY461(T) (=LMG 26031(T)=BCRC 80259(T)=KCTC 23308(T)).