In South Africa, serogroup B meningococcal disease is sporadic. The aim of this study was to characterize serogroup B strains causing invasive meningococcal disease (IMD) in South Africa from 2005 to 2008. Isolates, collected through a national, laboratory-based surveillance program for IMD, were characterized by multilocus sequence typing (MLST). Two thousand two hundred thirty-four cases were reported, of which 1,447 had viable isolates. Intermediate resistance to penicillin was observed in 2.8% (41/1,447) of all strains. Serogroup B was the second most common serogroup (17%, 251/1,447) and increased from 14% (58/414) in 2005 to 25% (72/290) in 2008 (P < 0.001); however, incidence remained stable during the study period (average incidence, 0.13/100,000 population) (P = 0.54). Serogroup B was predominantly characterized by three clonal complexes, namely, ST-41/44/lineage 3, ST-32/ET-5, and the new complex ST-4240/6688, which accounted for 27% (65/242), 23% (55/242), and 16% (38/242) of isolates, respectively. ST-4240/6688 was more prevalent among young children (<5 years) than other clonal complexes (27/37 [73%] versus 108/196 [55%]; P = 0.04). In the most densely populated province of South Africa, Gauteng, the prevalence of ST-32/ET-5 increased from 8% (2/24) in 2005 to 38% (9/24) in 2008 (P = 0.04). Capsular switching was observed in 8/242 (3%) strains. The newly assigned clonal complex ST-4240/6688 was more common in young children.