Streptococcus pneumoniae is both an aggressive pathogen and a normal part of the human respiratory microbiome. Clinicians and microbiologists have struggled to develop tests that can identify pneumococcal respiratory infection and accurately distinguish colonization from invasive disease. Molecular methods hold the promise of an improved ability to rapidly detect microorganisms in respiratory secretions and to make an accurate diagnosis; however, interpretation of diagnostic testing for S. pneumoniae remains problematic. Molecular assays, such as those targeting the pneumolysin gene, may cross-react with other streptococcal species, confounding detection and quantification. Assays that target the autolysin gene appear to be more specific. Even when accurately identified, however, the significance of S. pneumoniae DNA detected in clinical samples is difficult to determine. Here we will discuss the challenges faced in the interpretation of molecular testing for S. pneumoniae, and some strategies that might be used to improve our ability to diagnose pneumococcal respiratory infection.