Among viral diseases of animals, Foot and Mouth Disease is an exceedingly transferable disease of clovenhoofed animals caused by the virus of class Aphthovirus, family Picornaviridae. In the present study the Indirect Sandwich ELISA negative samples were tested through PCR and a range of OD for false negative ELISA samples was adjusted in order to decide whether the samples are actually positive or negative. Total 50 ELISA’s negative samples were confirmed through PCR. Out of total 50 ELISA’s negative samples, 23 were positive for PCR while rests of the 27 samples were found negative. In the PCR positive samples 13, 2 and 3 were positive for Serotype “O”, “A” and “Asia-1”, respectively. While 2 samples were positive for O and A and 3 for O and Asia-1 mixed infection. On the basis of type of samples, it was found that 18 out of 25 epithelial tissue samples and 5 out of 25 swab samples were positive. It was concluded that the ELISA negative samples with OD value 0.05 to 0.09 will be considered positive while the samples with OD value 0.04 or below should be considered negative. The epithelial tissues either from the oral commissar of foot region is the representative sample for diagnostic purpose.