Streptococcus pneumoniae (the pneumococcus) is a significant global pathogen. Accurate identification and serotyping are vital. In contrast with World Health Organization recommendations based on culture methods, we demonstrate that pneumococcal identification and serotyping with molecular methods are affected by sample type. Results from oropharyngeal samples from adults were often inaccurate. This is particularly important for assessment of vaccine impact using carriage studies, particularly in low- and middle-income countries where there are significant barriers for disease surveillance.