Foot-and-Mouth Disease is a vesicular disease of cloven-hoofed animals. This study was designed for Isolation and identification of FMDV type O from Ismailia governorate, the collected samples were 41 vesicular fluid and 41 tongue epithelial samples in the period between October 2013 to December 2014. Forty one pooled samples were inoculated in BHK-21 cell culture for isolation. 34 isolates out of 41 pooled samples showed maximum FMDV like CPE. Isolated FMDV were passed for 3 successive passage in BHK-21 cells. Last passage of each isolates was identified and confirmed 30 samples positive as FMDV type O by complement fixation test and PCR. All FMDV isolates give a suspected bands at 600bp when PCR product examined by agarose gel electrophoresis and UV illumination system. PCR products of three isolates were selected for nucleotide sequencing representing 2 buffaloes from El-Kassasin, Ein Ghosin and one cattle from Abou Sultan. Sequence of VP1 gene of the three isolates were submitted to the gene bank with accession number of (KT121465), (KT121466) and (KT121467). When the VP1 sequence of the current strains compared to other Egyptian and non Egyptian strains submitted to gene bank, it showed that great divergence ranged between 1.2 and 12.7 %, meanwhile, sequence comparison with O Sudan 5 2008 strain showed a great identity ranged between 93 to 94.2 % indicating that the FMDV type O viruses circulating in Ismailia may evolved from the FMDV circulating in Sudan (O Sudan 5 2008).