Detection of diarrheagenic Aeromonas specific aerolysin toxin (Aer) gene by PCR based assay and isolation, identification of diarrhea causing Aeromonas from faeces by culture methods were carried out in the Division of Active Surveillance, National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India for a period of 12 months. Out of 602 faecal samples collected from patients with acute diarrhea admitted in Infectious Diseases (ID) Hospital, Kolkata, 68 (11.29%) samples were found to be possessing Aer gene by PCR technique. The conventional culture methods using selective media yielded only 64 (10.6%) Aeromonas strains from the same faecal samples. The different Aeromonas species possessing Aer gene identified by PCR based technique include A. hydrophila (55.8%), A. caviae (17.6%), A. veronii (10.2%), A. schubertii (4.4%), A. jandaei (2.9%) and A. trota (8.8%). The isolation and identification of Aeromonas by routine culture did not detect enterotoxigenic A. trota present in four diarrheal faecal samples. The failure of the growth of enterotoxigenic A. trota on selective media may be attributed to the ampicillin susceptibility of those strains. The quality control studies revealed that PCR method for the direct detection of Aer gene from the faeces has the sensitivity of 100% and specificity of 98%.