This study reports the molecular characterization of foot-and-mouth disease virus (FMDV) in the provinces of Punjab and Sindh, Pakistan during 2014-17. FMDV genome was detected in 42 and 41 out of 46 samples (epithelial tissue and saliva) by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Sequences of the complete VP1 coding region of the samples (n = 33) was achieved showing that 10, 4 and 19 samples belonged to serotype O, A and Asia1 respectively. Phylogenetic analysis of serotype O revealed that at least one novel sublineage within the ME-SA topotype is circulating in the region, named here as PAK-14. This sublineage showed similarity with the viruses circulating in Turkey and Pakistan during 2010 indicating that viruses circulating in these countries have common origin. Analysis of serotype A viruses revealed a new lineage is circulating in the region, reported here as A-PAK14 showing close identity with the strain prevalent in Pakistan during 2007. Circulation of these new linages in the region shows continuous evolution of the viruses. Two of the undisclosed serotype A sublineages within the Iran-05 lineage were also found circulating in the region. In addition, molecular investigation of the VP1 coding region sequences of serotype Asia1 strains revealed that they belong to Group-VII (Sindh-08). Interestingly some of the serotype Asia1 isolates (n = 6) showed 99.9% similarity (among themselves) although they were collected from different districts more than 100 Km apart from one another. This unusual conservation among serotype Asia1 over long distances can be explored by studying the role of wild animals, slaughter houses and milk collection centres in the spread the disease.