Polymerase chain reaction (PCR) using theMycoplasma fermentansinsertion sequence-like element (ISLE) RW primer set amplifies DNA fromMycoplasma oralewhen more than 1 ng is present in the reaction tube. In this study, amplified products from 11 different clinical isolates and the ATCC prototype ofM. oralewere sequenced and compared to 206 bp amplicons from eight isolates and the ATCC strain ofM. fermentans. The nucleotide sequences of the amplifiedM. oraleproducts had high sequence homology (88–92%) to those fromM. fermentans, but differed at several key positions. TheM. oraleproducts contained a DraI restriction enzyme site not found in any of theM. fermentansamplified products. Consistent with this finding, the PCR products fromM. oralewere digested by DraI while the PCR products fromM. fermentanswere resistant to DraI digestion. The results suggest thatM. oralemay carry a similar IS-like element that complicates but does not negate using the ISLE PCR assay designed to detectM. fermentans. It appears possible for the RW primers to amplifyM. oraleif the mycoplasmas are present at higher concentrations. The amplified products can be differentiated from those fromM. fermentansby a rapid DraI restriction endonuclease digestion or by Southern blot analysis using the RW006 internal probe under highly stringent conditions.