Forty strains isolated from soil taken from a hay meadow were assigned to the genus Dactylosporangium on the basis of colonial properties. 16S rRNA gene sequence analysis showed that the isolates formed a group that was most closely related to the type strain of Dactylosporangium aurantiacum, but well separated from other Dactylosporangium type strains and from ‘Dactylosporangium salmoneum’ NRRL B-16294. Twelve of 13 representative isolates had identical 16S rRNA gene sequences and formed a subclade that was distinct from corresponding phyletic lines composed of the remaining isolate, strain BK63T, the ‘D. salmoneum’ strain and the type strainsof recognized Dactylosporangium species. DNA–DNA relatedness data indicated that representatives of the multi-membered 16S rRNA gene subclade, isolate BK63T and the ‘D. salmoneum’ subclade formed distinct genomic species; all of these organisms had chemotaxonomic and morphological properties consistent with their classification in the genus Dactylosporangium. They were also distinguished from one another and from the type strainsof recognized Dactylosporangium species based on a range of phenotypic properties. Combined genotypic and phenotypic data showed that isolate BK63T, isolates BK51T, BK53 and BK69, and strain NRRL B-16294T should be classified in the genus Dactylosporangium as representing novel species. The names proposed for these species are Dactylosporangium luridum sp. nov. (type strain BK63T = DSM 45324T = KACC 20933T = NRRL B-24775T), Dactylosporangium luteum sp. nov. (type strain BK51T = DSM 45323T = KACC 20899T = NRRL B-24774T) and Dactylosporangium salmoneum sp. nov., nom. rev. (type strain NRRL B-16294T = ATCC 31222T = DSM 43910T = JCM 3272T = NBRC 14103T).