The need for a microbial identification of Streptococcus pneumoniae independent of culture methods has resulted in the introduction of other laboratory principles. The verification of a proper and exclusive gene for the detection of the pneumococcus by nucleic acid-based tests is, however, still unresolved. A previously published lytA-gene-specific real-time PCR method was applied to a panel of bacterial strains to clarify the analytical sensitivity and specificity of a PCR assay targeting this gene. Furthermore, a phylogenetic analysis of published lytA gene sequences was performed to look at gene sequence differences and the theoretical match with the primers and probes. The lytA-gene-specific PCR detected 46/46 S. pneumoniae isolates. All 49 of the non-pneumococcal isolates tested negative, including 22 isolates from the mitis group streptococci. Phylogenetic analysis of 94 sequences of the lytA gene from different strains of S. pneumoniae, Streptococcus mitis and Streptococcus pseudopneumoniae showed that 70/87 S. pneumoniae sequences constituted one cluster and a further six sequences were outside but adjacent to this cluster, all with a complete match with primers and probes. The remaining 11 S. pneumoniae strains could be placed in a different cluster, which also contained the five S. mitis and two S. pseudopneumoniae strains. All strains had no match with primers and probes. The S. pneumoniae strains in the second cluster were all characterized by being bile-insoluble, an infrequent pneumococcal phenotype. Routine laboratories can utilize the additional observation that pneumococci that were negative by the specific PCR also carried the phenotype of bile insolubility, thereby observing the incidence of false-negative results produced by the PCR assay. The real-time PCR targeting the lytA gene thus constitutes a sensitive and specific assay that distinguishes S. pneumoniae from its close relatives in the mitis group.