ABSTRACTSmall-colony variants (SCVs) of bacteria are associated with recurrent and persistent infections. We describe for the first time SCVs ofStreptococcus tigurinusin a patient with a prosthetic joint infection.S. tigurinusis a novel pathogen of theStreptococcus mitisgroup and causes invasive infections. We sought to characterizeS. tigurinusSCVs using experimental methods and find possible genetic explanations for their phenotypes. TheS. tigurinusSCVs were compared with the wild-type (WT) isolate using phenotypic methods, including growth under different conditions, autolysis, and visualization of the cell ultrastructure by use of transmission electron microscopy (TEM). Furthermore, comparative genome analyses were performed. TheS. tigurinusSCVs displayed reduced growth compared to the WT and showed either a very stable or a fluctuating SCV phenotype. TEM analyses revealed major alterations in cell separation and morphological abnormalities, which were partially explained by impaired autolytic behavior. Intriguingly, the SCVs were more resistant to induced autolysis. Whole-genome sequencing revealed mutations in the genes involved in general cell metabolism, cell division, stringent response, and virulence. Clinically, the patient recovered after a 2-stage exchange of the prosthesis. Comparative whole-genome sequencing in clinical strains is a useful tool for identifying novel genetic signatures leading to the most persistent bacterial forms. The detection of viridans streptococcal SCVs is challenging in a clinical laboratory due to the small colony size. Thus, it is of major clinical importance for microbiologists and clinicians to be aware of viridans streptococcal SCVs, such as those ofS. tigurinus, which lead to difficult-to-treat infections.