BACKGROUND: Streptococcus pneumoniae serotype 5 is among the most common serotypes causing invasive pneumococcal disease (IPD) in The Gambia. We anticipate that introduction of the 13-valent pneumococcal conjugate vaccine (PCV-13) into routine vaccination in The Gambia will reduce serotype 5 IPD. However, the emergence of new clones that have altered their genetic repertoire through capsular switching or genetic recombination after vaccination with PCV-13 poses a threat to this public health effort. In order to monitor for potential genetic changes post-PCV-13 vaccination, we established the baseline population structure, epidemiology, and antibiotic resistance patterns of serotype 5 before the introduction of PCV-13. METHODS: Fifty-five invasive S. pneumoniae serotype 5 isolates were recovered from January 2009 to August 2011 in a population-based study in the Upper River Region of The Gambia. Serotyping was done by latex agglutination and confirmed by serotype-specific Polymerase Chain Reaction (PCR). Genotyping was undertaken using Multilocus Sequence Typing (MLST). Antimicrobial sensitivity was done using disc diffusion. Contingency table analyses were conducted using Pearson's Chi(2) and Fisher's exact test. Clustering was performed using Bionumerics version 6.5. RESULTS: MLST resolved S. pneumoniae serotype 5 isolates into 3 sequence types (ST), namely ST 289(6/55), ST 3339(19/55) and ST 3404(30/55). ST 289 was identified as the major clonal complex. ST 3339, the prevalent genotype in 2009 [84.6% (11/13)], was replaced by ST 3404 [70.4% (19/27)] in 2010 as the dominant ST. Interestingly, ST 3404 showed lower resistance to tetracycline and oxacillin (P < 0.001), an empirical surrogate to penicillin in The Gambia. CONCLUSIONS: There has been an emergence of ST 3404 in The Gambia prior to the introduction of PCV-13. Our findings provide important background data for future assessment of the impact of PCV-13 into routine immunization in developing countries, such as The Gambia.