Outbreaks of Foot and Mouth Disease (FMD) cause severe economic loss to the livestock industries in terms of loss of meat, milk production and the high mortality rateespecially in calves. This study was intended for detection, isolation and molecular characterization of FMDV circulated strain among different regions in Beni-Suef governorate, Egypt during 2015 and 2017. One hundred field samples were collected from clinically diseased cattle and buffaloes including vesicular fluid and sloughed epithelial membrane. Molecular detection and differentiation of FMDV serotypes by RT-PCR showed that (71%) were positive for serotype O indicating its predominance in Beni-Suef, Egypt.Also (18%) were positive for serotype SAT2 and (7%) were un-typed serotype being only positive for universal primer. Twenty samples were isolated on BHK-21 cells clone 13. Three samples showed the characteristic CPE of FMDV after blind passage 4 times. Sequences of VP1 coding-region of the three isolated FMD virus showed that the three isolated viruses were serotype O. The phylogenetic analysis of the isolated viruses reveled that they were closely related to type O of those reported in Ismailia, Behira, Giza and Cairo during 2016 with identity ranged from 97.7% - 99.8%. The most relevant outboard isolate was SUD/8/2008 with 93% identity after Blast homology search. However, the phylogenetic tree analysis revealed that the three FMDV type O isolates FMDV/serotype O/Beni-Suef/2017 differs partially from all other Egyptian serotypes of 2016. In conclusion, serotype O was the most prevalent FMDV serotype in Beni-Suef, Egypt.