Background: Foot-and-mouth disease (FMD) is one of the contagious and the most economically devastating viral diseases caused by the FMD virus. The present study was conducted with the aim of molecular characterization of the FMD virus isolated from outbreaks that occurred in cattle in two different districts of Amhara regional state of Ethiopia. Samples collected from outbreaks were isolated on BHK-21 cell and serotype were detected using antigen detection ELISA. Phylogenetic and amino acid variability analyses of isolated viruses were undertaken after sequencing the VP1 gene in World Reference Laboratory, Pirbright, UK.Result: In the present study only serotypes O was found in the samples analysed using antigen detection ELISA and in 10 out of 13 (76.9%) cultured samples, virus were isolated on BHK-21 cells. Phylogenetic analysis of these isolates revealed that, they belonged to East Africa topotype-3 (EA-3) and shared 96.6% nucleotide similarity with Sudan’s isolates. A total of 10.3% amino acid variations were recorded between VP1 gene sequences of the field isolates of serotype O FMDV and vaccine strain (O/ETH/38/2005) used in Ethiopia for vacccine production.Conclusion: The phylogenetic analysis serotype O detected in this study revealed that the virus was clustered with East African topotype-3 (EA-3) and exhibited high genetic similarity with isolates from Sudan. A number of amino acid variations were also noted at different sites of VP1 gene when comparing field isolates with the vaccine strain. Thus, to enhance control of FMD in Ethiopia, detailed molecular analysis of the field isolates along with in-vitro vaccine matching tests need to be undertaken at frequent intervals to assess the protective potential of the vaccine strain in use.