In this study, we developed a simple, reliable, serotype-specific PCR method to differentiate Streptococcus pneumoniae serotypes 6A, 6B, and 6C. It was more efficient and practical than the assays currently being used to identify serotypes 6A, 6B, and 6C. Of 120 selected serogroup 6 isolates from subjects with invasive (n = 101) and noninvasive (n = 19) pneumococcal disease, most of which were collected after 2003 in New South Wales, 45 had been identified as 6A and 75 had been identified as 6B by the Quellung reaction. PCR analysis confirmed the results for serotype 6B isolates and identified two different subtypes. Fourteen of 45 isolates that had been identified as serotype 6A actually belonged to serotype 6C.