Two novel bacterial strains were isolated from tropical rain forest soil from Thailand. Strains A-T 0875(T) and A-T 1383(T) stained Gram-positive and were filamentous bacteria that developed cylindrical sporangia containing four oval- to rod-shaped spores at the ends of short sporangiophores on branched aerial mycelium. The cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid and alanine as cell-wall amino acids; whole-cell hydrolysates contained rhamnose, madurose, glucose, galactose and 3-O-methylmannose as whole-cell sugars. The predominant menaquinone was MK-9(H(4)). Mycolic acids were not detected. The diagnostic phospholipid was phosphatidylethanolamine. The predominant cellular fatty acids were iso-C(16 : 0) and 10-methyl-C(17 : 0). For both strains, the G+C content of the genomic DNA was 71 mol%. Phenotypic and chemotaxonomic analyses showed that the characteristics of the two isolates were typical of members of the genus Planotetraspora. Furthermore, 16S rRNA gene sequence analysis also indicated that the strains belonged to the genus Planotetraspora but as representatives of two novel species. Following an evaluation of our phenotypic, chemotaxonomic and genotypic studies, two novel species are proposed, Planotetraspora kaengkrachanensis sp. nov. (type strain A-T 0875(T)=BCC 24832(T)=NBRC 104272(T)) and Planotetraspora phitsanulokensis sp. nov. (type strain A-T 1383(T)=BCC 26045(T)=NBRC 104273(T)).