Entamoeba gingivalisis a parasitic protist that resides in the oral cavity. AlthoughE. gingivalishas been frequently detected in individuals with periodontitis, its precise role in this context remains to be established, sinceE. gingivalisis also regularly found in healthy individuals. Sequence data onE. gingivalisare still scarce, with only a limited number of sequences available in public databases. In this study, a diagnostic PCR protocol was established in order to obtain a first impression on the prevalence ofE. gingivalisin Austria and enable a differentiation of isolates by targeting the variable internal transcribed spacer regions. In total, 59 voluntary participants were screened forE. gingivalisand almost 50% of the participants were positive, with a significantly higher prevalence of participants with self-reported gingivitis. Moreover, in addition to the established subtypes ST1 and ST2, a potentially new subtype was found, designated ST3. 18S DNA sequencing and phylogenetic analyses clearly supported a separate position of ST3. Interestingly, subtype-specific PCRs revealed that, in contrast to ST2, ST3 only occurred in association with ST1. ST2 and ST1/ST3 were more often associated with gingivitis; however, more data will be necessary to corroborate this observation.