Motile Aeromonas Septicaemia (MAS) is a common fresh water fish disease and major threat to the aquaculture in Pakistan. The present study was carried out on suspected cases of Rohu (Labeo rohita) to characterize and genetically analyze local strains of Aeromonas (A.) hydrophila, a key pathogen responsible for the said disease in farmed fish of Pakistan. A total of ninety suspected samples were collected from fish farms in various districts of Punjab from June 2018 to April 2019. The samples were processed and A. hydrophila strains were isolated. The primary identification of sixty seven isolates was verified by colony morphology, microscopy and phenotypic characterization with ten biochemical reactions. The A. hydrophila strains of test samples were characterized by polymerase chain reaction (PCR) using 16S rRNA at desired size of 356 bp. The PCR amplified product was subjected to DNA sequencing and phylogenetic analysis showed homology with related strains of Aeromonas spp. By antibiotic sensitivity test, the isolates were checked for nine antibiotics, in which pathogen was sensitive to four and resistant to five drugs. Results of molecular characterization confirmed strains as A. hydrophila which are useful to take preventive measures against the said disease.