Here, we report a laboratory-based study of Streptococcus pneumoniae recovered from patients with meningitis in Rio de Janeiro State, Brazil. The aim of this study was to determine the evolution of β-lactam resistance, antimicrobial susceptibility pattern, serotypes, and genetic diversity of S. pneumoniae, isolated from meningitis patients between 2000 and 2008. A total of 264 S. pneumoniae recovered from patients between 2000 and 2008 were included. Susceptibility testing (E-test) of S. pneumoniae showed resistance to penicillin, ceftriaxone, oxacillin, cotrimoxazole, tetracycline, ofloxacin, erythromycin, chloramphenicol, and rifampicin. Penicillin resistance (PEN-R, minimal inhibitory concentration [MIC] ≥ 0.12 μg/mL) increased from 8% of isolates in 2000-2002, to 12% in 2003-2005, and to 20% in 2006-2008. Ceftriaxone resistance (MIC ≥ 1.0 μg/mL) was detected among some PEN-R isolates (13%) from 2004 onward. Within the PEN-R isolates, serotypes that are included in 10-valent pneumococcal conjugate vaccine predominated (90%), and resistance was detected mostly in isolates of serotypes 14 (61%), 23F (16%), 6B (10%), and 19F (3%). Multilocus sequence typing showed that 52% of the PEN-R isolates, and 89% of those with MICs ≥ 0.5 μg/mL, were sequence type (ST)-156 or single-locus variants of this ST (ST-557 or ST-4388); all of these isolates were serotype 14 and were assigned to the Spain-3 clone. β-lactam resistance increased recently among cerebrospinal fluid isolates and was mainly due to the surge of the ST-4388, a previously undescribed gki single-locus variants of ST-156. Regional surveillance is shown to be essential to provide optimal antimicrobial therapy, monitor highly successful clones, and formulate adequate vaccination strategy.