Serogroup 6 of Streptococcus pneumoniae contains four established serotypes (6A-6D). Recently, putative serotype 6E (genotype 6E) was proposed as a novel type, which is cross-reactive with 6B-specific antiserum, but its capsular polysaccharide synthesis (cps) locus is genetically distinct from those of serotypes 6A and 6B. In the present study, prevalence of genotype 6E was analyzed by a newly designed multiplex polymerase chain reaction (PCR) for noninvasive or colonizing S. pneumoniae isolates in northern Japan assigned to serogroup 6 in our previous study by the sequential multiplex PCR developed by Pai et al. Among the isolates previously assigned to 6A and 6B, 2.2% (1/45) and 77.3% (140/181) of isolates, respectively, were revealed to have cps genes of genotype 6E. Eight 6E isolates selected for further analysis were found to have identical or highly similar sequences of cps genes (wzg, wzh, wze, wciN, wciP, and wzy) to those of strains previously reported as putative serotype 6E, and all the isolates were classified into sequence type 90 (ST90). Reanalysis of genetic traits on penicillin and macrolide resistance clarified significantly higher rates of three pbp mutations (gPRSP) and ermB in genotype 6E than in serotypes 6A and 6B. These findings suggested a need for detection of genotype 6E in the surveillance of S. pneumoniae serotypes.