We describe here the use of real-time DNA sequence analysis of Helicobacter pylori 16S rRNA gene fragments by pyrosequencing for rapid molecular identification and subtyping of clinical isolates based on DNA sequence heterogeneity within the variable V1 and V3 regions. Six individual 16S rDNA V1 alleles (position 75-100) were identified in 23 clinical isolates obtained from gastric biopsy specimens. Eleven of these revealed sequence identities with H. pylori 26695 and one was identical with the rrn genes in strain J99. The other V1 alleles showing single or double nucleotide mutations or single nucleotide insertions could be divided into four groups with 5, 4, 1, and 1 isolates each. Two out of 25 isolates demonstrated single C to T transitions in the V3 region (position 990-1020). The present findings show that subtle DNA sequence variation occurs sufficiently often in the 16S rDNA variable V1 and V3 regions of H. pylori to provide a consistent system for subtyping.