Present research investigation aims to investigate the prevalence of Clostridium perfringens type A in fresh water fishes [common carp, cat fish, silver carp] obtained from randomly selected dams, freshwater lakes, ponds, retail shops and local markets of Tamil Nadu, India. Intestinal contents from 267 fishes were processed for the isolation and identification of C. perfringens. The ultra structures of the bacterial isolates were carried out in Scanning electron micrograph [SEM] and their virulence determinantion was done by Polymerase chain reaction [PCR]. Bacteriological investigation revealed the presence of C. perfringens in 49 [18.35%] samples. The ultrastructure studies of the bacterial isolates in SEM were observed to be in clusters of thick rods with variable in length and mostly occurred in pairs. In PCR, out of four major virulence genes of C. perfringens, alpha toxin gene [cpa] was harboured by all the isolates. Along with cpa, additional beta2 toxin gene [cpb2] was also detected in 29 [59.18%] isolates of C. perfringens. But none of the isolates were found positive for other major virulence genes [beta, epsilon and iota] and enterotoxin genes. This PCR determination of virulence genes suggested that the isolated C. perfringens from fresh water fish samples were belonged to type A.