Burkhloderia pseudomallei is the causative agent of melioidosis, a disease in which treatment failures and relapses are common. This study reports on slow growing B. pseudomallei 'small colony variants' (SCVs), isolated either in vitro after exposure to ceftazidime, ciprofloxacin or gentamicin or from the spleen and liver in a mouse model of melioidosis after treatment with ceftazidime. Interestingly, SCVs isolated by either method or antimicrobial agent showed a significant increase in the minimal inhibitory concentrations of various unrelated classes of antimicrobial agents. B. pseudomallei SCVs did not differ from their parental strains in standard biochemical profiles, nor by pulsed field gel electrophoresis or electron microscopy. Although the SCV phenotype was stable throughout numerous passages on antibiotic-free solid media, revertants with the parental colony morphology and, most importantly, with the parental susceptibility pattern occurred. These revertants led to rapid overgrowth of SCVs in liquid media without added antibiotics. Future studies will have to determine the clinical relevance of B. pseudomallei SCVs especially in treatment failure and relapse of infection.