Until recently, group B streptococcus, serotype V (GBS-V), was an infrequent cause of disease. It is now recognized as a significant cause of infections in both children and adults. To determine if this increase was due to the recent introduction and spread of a single clone of GBS-V, we analyzed, by pulsed-field gel electrophoresis (PFGE), the SmaI chromosomal DNA digests of 45 bacteria: 41 isolated from human infections between 1986 and 1996 in the United States, 2 from human infections in Argentina, and 2 from naturally infected mice. Seventeen patterns were found and arbitrarily designated patterns A to Q. Pattern N constituted 24 (53%) of the isolates and was found in all of the years tested and from all surveillance areas, as well as in both isolates from Argentina, and was very similar to the GBS-V isolated from a mouse. Pattern P was found in three isolates, pattern F was found in two, and the remaining patterns were found in one isolate each. We concluded that the majority of isolates of GBS-V are of one PFGE subtype and that this subtype was predominate before the increase in disease caused by GBS-V and that GBS-V disease is caused by several different subtypes.