Kitasatosporia strains were found to be resistant to an antibiotic, novobiocin. By a new method using novobiocin-containing agar medium, five Kitasatosporia strains, SK-3255, OM-5023, SK-3406, SK-3412 and SK-3421 were efficiently isolated from soils. These five strains were characterized in common by Rectiflexibiles, spores cylindrical with smooth surfaces, and no melanin formation. Two strains, SK-3255 and OM-5023 were taxonomically different from known species of the genus Kitasatosporia, while the two strains, SK-3406 and SK-3412 and the remaining strains SK-3421 were identified with K. phosalacinea and K. papulosa, respectively.