To investigate the clinical and microbiological characteristics of infections caused by Tsukamurella spp., the computerised database of the Bacteriology Laboratory at National Taiwan University Hospital (Taipei, Taiwan) was reviewed retrospectively to identify patients with infections caused by this species during the period January 1997 to December 2008. All of the isolates had been initially misidentified as Rhodococcus spp. Identification of Tsukamurella isolates to species level was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the heat shock protein gene (hsp65) as well as 16S rRNA gene sequencing. During the study period, a total of eight patients with Tsukamurella infection and two patients with Tsukamurella colonisation were identified. Tsukamurella tyrosinosolvens (n=6) was the most prevalent species, followed by Tsukamurella spumae (n=3) and Tsukamurella pulmonis (n=1). Keratitis was the most common type of infection (n=3), followed by catheter-related bloodstream infection (n=2). One of the patients with Tsukamurella infection died due to bacteraemia; the other seven patients with Tsukamurella infection had favourable outcomes. The three species had different drug susceptibility patterns; T. pulmonis was the most resistant pathogen, with higher minimum inhibitory concentrations of clindamycin (>2 mg/L), erythromycin (2 mg/L) and tetracycline (8 mg/L) than those for the other Tsukamurella spp. In conclusion, strains of Tsukamurella spp., including T. spumae, are uncommon causative agents of ocular infections and bacteraemia in cancer patients. Molecular diagnostic methods are essential to distinguish species in the Tsukamurella genus from species in other phylogenetically related genera such as Rhodococcus.