Objectives: Stenotrophomonas maltophilia is a recently identified nosocomial pathogen in Malaysia. Despite limited pathogenicity, its rate of isolation has increased in recent years. The aim of this study was to investigate the antibiotic susceptibility patterns, antibiotic resistance determinants, and the epidemiology of S. maltophilia at the largest tertiary care hospital in Malaysia. Methods: This study was carried out from January to December 2008. Sixty-four S. maltophilia isolates were investigated for their antibiotic susceptibility patterns by disk diffusion test and E-test. The antibiotic resistance mechanism for trimethoprim-sulfamethoxazole (TMP-SMX) was assessed by PCR for sul1, sul2, qac/smr, and class 1 integrons in general. Epidemiological relatedness among isolates was determined by pulsed-field gel electrophoresis (PFGE). Results: The highest number of S. maltophilia infections was observed in the intensive care unit (ICU) (n = 13; 20.3%), while the lowest number of infections was seen in the neurology, psychiatric, and dermatology wards (each n = 1; 1.6%). All isolates were susceptible to minocycline. One isolate was resistant to TMP-SMX with a minimum inhibitory concentration (E-test) > 32 mg/l. The strain carried the sul1 gene and class 1 integron. None of the isolates were positive for the qac/smr genes. Alth Conclusion: The increasing number of S. maltophilia isolates seen in the ICU, their resistance to mainstay antibiotics, their genetically diverse nature, and possible cross-transmission within the hospital, strongly underscores the need for continuous surveillance for S. maltophilia in the hospital setting. (C) 2012 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.