Foot and mouth disease is caused by Apthovirus, family Picornaviridae. In this study, RT-PCR base detection of FMDV for primary and serotype specific diagnosis was developed and sequence analysis was done for molecular epidemiology. Different types of clinically positive samples of FMD were collected from the field which includes swab samples from the mouth and hoff vesicles, serum, plasma, saliva and milk samples from cattle, buffalo and goats. RT-PCR based identification and optimization was done with universal primer sets (P1 / P2 and F1 / F2) with expected band of 216bps and 328bps. PCR optimization was done for serotype specific primers on vaccine and known positive serotypes. Nested PCR was optimized and performed to confirm the ‘O’ serotype of FMD. Cloning of PCR product of representative samples were done into PTZ 57 R vector for sequencing. These sequences were mapped onto serotype specific dendrogram. It showed that the ‘O’ serotype circulating in Pakistan was very diverse genetically from the ‘O’ serotype in Middle East and Europe respectively and very much similar genetically to its neighbour countries (Sri Lanka, India, Iran, Iraq and China etc).