Group B streptococci (GBS) are serotyped according to capsular polysaccharide (CPS) type (Ia to VIII); an isolate is classified as nontypeable (NT) if no detectable CPS is found. Surface-localized protein antigens (α, β, R1, and R4) serve as additional markers to classify GBS isolates, which is particularly useful since NT isolates often express one or more of these proteins. To compare genetic resemblance among isolates with similar protein profiles, we studied 58 NT isolates digested with the SmaI macrorestriction enzyme prior to pulsed-field gel electrophoresis (PFGE). Of these 58, 15.5% expressed α only, 20.7% expressed α+β, 15.5% expressed R4, and 25.8% expressed R1,R4, while 22.4% of the isolates expressed no detectable proteins. The largest PFGE profile group, with 48% of the isolates, was group 4, composed primarily of isolates that expressed R1,R4 or no proteins. The second most common profiles were 3 and 32, each with 13.8% of the isolates. Since NT isolates in profile group 4 were highly related to type V isolates, as demonstrated by PFGE profiles, we investigated 45 type V isolates. Two-thirds of the type V isolates within profile group 4 were classified into subgroup 4a, compared to 28.2% of 39 NT isolates. Only 11% of the V/R1,R4 isolates were identical to the prototype group 4 profile, in contrast to 75% of the NT/R1,R4 isolates. A shift of type V isolates into profile 4 subgroups may be indicative of a genetic change over time. PFGE is a valuable approach for comparison of GBS isolate relatedness and for monitoring of NT and typeable GBS isolates for potential clonal divergence.