Aims: Present research is focused on the molecular level characterization of drug-resistant Listeria monocytogenes identified from food and water samples from Tamil Nadu, India. Methodology and results: A total of 39 food and water samples were collected from local markets and retail shops in Tamil Nadu, India and processed for the isolation and identification of bacteria. Morphology of the bacteria was analysed under a fluorescent microscope. Isolated bacteria were serotyped and screened for the presence of virulence-associated genes haemolysin (hlyA) and invasive associated protein (iapA) by Real-time polymerase chain reaction. The qPCR positive isolates were also typed by random amplified polymorphic DNA-PCR for epidemiological study. Antibiotic resistance test was done with 16 commercial antibiotics by disc diffusion method. A total of 8 (20.51%) L. monocytogenes were identified belonging to the serotype group 1/2a, 1/2b, 1/2c and 4b. PCR assays revealed the presence of hlyA (456 bp) and iapA (131 bp) genes. In RAPD, OPA-10 primer was found to generate the distinct polymorphic fragment among the isolates. All the isolates were 100% resistant to rifampicin, co-methoxazole, linezolid and oxacillin and 100% sensitive to tetracycline and chloramphenicol. Tetracycline and chloramphenicol are suggested to be a very effective antibiotic against the tested L. monocytogenes isolates. Conclusion, significance and impact of study The hlyA and iapA based quantitative PCR technique could be a rapid molecular technique for the detection of L. monocytogenes used in this study. Serotyping along with RAPD-PCR was able to discriminate between the isolates and therefore could serve as a robust and sensitive tool for typing antibiotic-resistant strains of L. monocytogenes.