We applied a set of commonly used universal primers (primers RW01 and DG74) to amplify partial fragments of 16S ribosomal DNA for bacterial identification and found an unexpected amplicon (547 bp), in addition to the expected 362-bp product, in samples containing Bacteroides thetaiotaomicron. It was demonstrated that the internal sequence (508 bp, excluding the primers) of the 547-bp amplicon was identical to the genomic sequence from nucleotide positions 165800 to 166307 of B. thetaiotaomicron type strain VPI-5482 by a BLAST search of the sequences in the GenBank database. The existence of this unexpected yet specific amplicon strongly indicated the presence of B. thetaiotaomicron in the sample, and it was found that it could be used to discriminate B. thetaiotaomicron from closely related species. Another set of PCR primers specific for B. thetaiotaomicron was developed on the basis of the sequence of this 547-bp genomic fragment. Both PCR-based assays showed the same sensitivity (88%) and specificity (100%).