Several methods have been used for typing of Streptococcus agalactiae (group B streptococci [GBS]). Methods currently in use may provide inadequate resolution (e.g., typing of capsular polysaccharides and surface protein) or are labor-intensive and expensive (e.g., multilocus sequence typing [MLST] or pulsed-field gel electrophoresis). This work describes the construction and use of a multiple-locus variant-repeat assay (MLVA) on 126 well-characterized human GBS strains, consisting mostly of invasive Norwegian strains and international reference strains. Based on in silico whole-genomic analysis of the genomes of strains A909, NEM316, and 2603V/R, 18 candidate loci were selected and investigated by PCR. Eleven loci showed diversity, and the five most diverse loci were used for the construction of an MLVA, consisting of a multiplex PCR followed by fragment analysis with capillary electrophoresis. The assay generated clusters which corresponded well with those observed by other methods. However, it provided a considerably higher degree of diversity, with 70 different MLVA types compared to 36 types generated by MLST. Simpson's index of diversity for the 5-locus MLVA was 0.963, compared to 0.899 for the MLST in this strain collection. MLVA results will generally be available within 2 days, which is usually faster than MLST. In our hands, MLVA of GBS represents a rapid, easy, and comparably inexpensive method for high-resolution genotyping of GBS.