Streptococcus bovis is being recognised increasingly as a cause of infective endocarditis, and has also been associated with underlying gastrointestinal malignancy. This study evaluated the molecular epidemiology of S. bovis isolates responsible for endocarditis or bacteraemia in Italian patients between January 1990 and August 2003. S. bovis isolates were classified on the basis of their biochemical profiles, antimicrobial susceptibilities and genotypes. Of 25 isolates studied, 20 were S. bovis I and five were S. bovis II. Seven biochemical profiles were identified. Pulsed-field gel electrophoresis (PFGE) analysis identified 22 profiles that differed by at least two DNA fragments and showed a similarity of < 87%. Most PFGE patterns represented single isolates that differed in antimicrobial susceptibility, but three PFGE types were observed, with identical profiles and antibiotypes, in isolates from two different patients. S. bovis I and II isolates grouped into two distinct genetic clusters (I and II) with a similarity coefficient of 38%. Two sub-clusters (Ia and Ib), with a similarity coefficient of 47%, included 17 S. bovis I isolates with similar biochemical profiles (15 with biotype A, and two with biotype B), but different resistance phenotypes. Based on the phenotypic and genotypic heterogeneity of the isolates, it is postulated that the increase in S. bovis endocarditis in this geographical area might have been caused by the selection of sporadic endemic clones from the endogenous intestinal flora.