Three bacterial strains, HKU51T, HKU52T and HKU53, were isolated from a conjunctival swab, corneal scraping and blood culture of three patients in Hong Kong with conjunctivitis, keratitis and catheter-related bacteraemia, respectively. Cells were Gram-stain-positive, aerobic, catalase-positive, non-sporulating and non-motile bacilli. The three strains had unique biochemical profiles that were distinguishable from those of closely related species of the genus Tsukamurella. Fatty acids, mycolic acids, cell-wall sugars and peptidoglycan analyses showed that they were typical of members of Tsukamurella. 16S rRNA gene sequence analysis revealed 100 % sequence identity between HKU52T and HKU53, and the two strains shared 99.5 % sequence identity with Tsukamurella sunchonensis JCM 15929T and Tsukamurella pseudospumae JCM 13375T; HKU51T shared 99.6 % sequence identity with Tsukamurella pulmonis CCUG 35732T. The DNA G+C contents of strains HKU51T, HKU52T and HKU53 were 70.9 ± 2.2, 71.3 ± 2.1 and 71.2 ± 2.3 mol% (mean ± sd; n = 3), respectively. DNA-DNA hybridization confirmed that the novel strains were distinct from other known species of the genus Tsukamurella ( ≤ 50.1 ± 3.7 % DNA-DNA relatedness); two of the isolates, HKU52T and HKU53, represented the same species ( ≥ 94.6 ± 5.6 % DNA-DNA relatedness), while the third isolate, HKU51T, represented another species. The novel species Tsukamurella hongkongensis sp. nov. is proposed to accommodate strains HKU52T and HKU53, with HKU52T ( = JCM 30715T = DSM 100208T) as the type strain; whilst another novel species, Tsukamurella sinensis sp. nov., is proposed to accommodate the third isolate, HKU51T ( = JCM 30714T = DSM 100207T), which is designated the type strain.