Foot and mouth disease (FMD) is an extremely contagious transboundary disease that affects cloven-hoofed animals. An uncontrolled animal movement has a significant role in the disease circulation in endemic areas. An accurate and early diagnosis is critical for FMD control. Seventeen tongue epithelium and three vesicular fluid samples were collected from five cattle farms, and nine tongue epithelium and one vesicular fluid samples were collected from three buffalo farms suspected to be infected with FMDV at Qalyubia government in 2021. Trail of virus isolation was carried out on BHK-21 cell line followed by conventional RT-PCR for typing of the obtained virus isolate. Partial sequencing and phylogenetic analysis of VP1 for the field isolate revealed that it was serotype O topotype EA-3 lineage Alx-17 (FMDV/O/EGY/Qalyubia/2021/OM681353) and closely related to the previously local Egyptian isolate in 2017 (FMDV/O/EGY/2017/OM221230.1) with nucleotide identity 99.36% and sharing nucleotide similarity 99.72% with the previously isolated virus from Sudan (FMDV/O/SUD/2017/MK422569.1) and Ethiopia (FMDV/O/ETH/2017/ MN987453.1) during 2017 that suggested the transboundary incursion. The isolated FMDV/O/EA-3/Alx-17 shared 85.65% nucleotide identity with the sequenced FMDV serotype O topotype ME-SA lineage Sharqaui-72 (FMDV/O/EGY/OM681355), while 15.28% nucleotide difference between the isolated FMDV/O/EA-3/Alx-17 and the vaccine strain Pan-Asia-2 (FMDV/O/ME-SA/Pan-Asia-2/OK642671.1). The sequenced Sharqaui-72 virus revealed 86.01% sequence identity with the Pan-Asia-2 vaccine strain. The authors recommend for periodical molecular and genetic characterization between field isolates and vaccine strains, more cross-matching (R-value) and challenge studies between EA-3 viruses and PanAsia-2 vaccine strain, in addition to strict quarantine measures for the imported animals