The high genetic similarity between some carboxydotrophic bacteria does not allow for the use of common sequencing techniques targeting the 16S rRNA gene for species identification. 16S rRNA sequencing fails to discriminate among Clostridium ljungdahlii and ‘Clostridium autoethanogenum’, despite this two species exhibit significant differences in CO2 assimilation and alcohol production. In this work we designed PCR primers targeting for the DNA gyrase subunit A (gyrA) and a putative formate/nitrite transporter (fnt) to specifically detect the presence of ‘C. autoethanogenum’, C. ljungdahlii or Clostridium carboxidivorans. We could confirm the simultaneous presence of C. ljungdahlii and ‘C. autoethanogenum’ in different bioreactors, and a preference of the latter for high CO2 content.