Foot-and-Mouth Disease (FMD) hinders the growth of the livestock industry in endemic countries like Bangladesh. The management and prevention of FMD are severely impacted by the high mutation rate and subsequent frequent generation of newer genotypes of the causative agent, Foot-and-Mouth Disease Virus (FMDV). The current study was conducted in nine districts of Bangladesh during 2019–21 to characterize the circulating FMDV strains based on the VP1 sequence analysis, the major antigenic recognition site providing serotype specificity and high variability of FMDV. This study detected the first emergence of the SA-2018 lineage in Bangladesh along with the predominance of Ind-2001e (or Ind-2001BD1) sublineage of ME-SA topotype under serotype O during 2019–21. The mutational spectrum, evolutionary divergence analysis and multidimensional plotting confirmed the isolates collected from Mymensingh districts, designated as MYMBD21 as a novel sublineage under the SA-2018 lineage. Analysis of the amino acid sequence revealed several changes in the G-H loop, B-C loop and C-terminal region of VP1, revealing a 12–13% divergence from the existing vaccine strains and a 95% VP1 protein homology, with most of the mutations potentially considerable as vaccine escape mutations, evidenced by three-dimensional structural analysis. This is the first report on the emergence of the SA-2018 lineage of ME-SA topotype of FMDV serotype O in Bangladesh, as well as a possible mutational trend towards the emergence of a distinct sublineage under SA-2018 lineage, which calls for in-depth genome-wide analysis and monitoring of the FMD situation in the country to implement a strategic vaccination and effective FMD control program.