Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes heavy economic losses. The causative agent, foot-and-mouth disease virus (FMDV) exists in seven distinct serotypes i.e. O, A, C, Asia-1, SAT1, SAT2 and SAT3. Multiple subtypes can also be identified within these serotypes. The present study reports the distribution of FMDV in Pakistan during the period 1952 to 2007. During this time, 1543 out of 2484 epithelial samples from suspect cases of FMD were found positive. Serotype O was the most prevalent followed by Asia-1 and A. The disease was more prevalent (P<0.001) in cattle than buffaloes. Higher numbers of outbreaks of the disease occurred between January to March during 2002 to 2007, which may result from livestock movement due to the festival, Eidul Azha, in which animals are sacrificed. Some 1501 oral swab samples from Pakistan, Afghanistan and Tajikistan were collected from clinically healthy animals between July, 2008 and August, 2009. RNA was extracted from the samples and was subjected to real time RT-PCR for detection of FMD viral genome. In addition, RNA was also extracted from 142 epithelial samples collected from clinically suspect cases of FMD between 2005-2009. Samples with Ct values of ≤30 were further processed for sequencing the whole VP1 coding region to identify the serotype and sub-type of the virus. Nucleotide sequences were also obtained from GenBank. Sequence comparisons were performed to establish the phylogenetic relationships between the viruses. The samples from two (of four) animal markets in Pakistan, one of three markets in Afghanistan and both the live animal markets in Tajikistan all tested negative. However, ~2% of samples from Gondal and 9% from Chichawatni in Pakistan were positive for FMDV RNA. Similarly, 15% of samples from Kabul and 50% from Badakhshan in Afghanistan were found positive. Serotypes A and O of FMDV were identified within these samples. In addition, oral swab samples were collected from dairy colonies in Lahore and Nagori (Karachi) but all tested negative. In the Landhi dairy colony, a cohort of 179 apparently healthy animals was studied. On their arrival, 22% of these animals were found positive for FMDV RNA (serotype A was identified) while 73% had antibodies to FMDV non-structural proteins. Thus newly introduced animals may be a significant source of the disease in the colony. Nucleotide sequences encoding at least the complete VP1 protein for 122 FMDVs from Pakistan and Afghanistan were determined. Phylogenetic analysis of the serotype O FMDVs present between 1997 and 2009 revealed the presence of multiple lineages within the ME-SA (Middle East South Asia) topotype. The PanAsia lineage is currently dominant and has evolved into distinct variants e.g. PanAsia-II and PanAsia-III. The rates of evolution of the O-PanAsia-II and III sublineages were 6.65 × 10-3 and 7.80 × 10-3 substitutions per nucleotide per year (s/nt/yr), respectively. Genetic analysis of serotype A FMDV from these countries collected between 2002 and 2009 revealed the presence of at least four lineages within two genotypes in the Asia topotype. The predominant lineage was A-Iran05 which has evolved into seven distinct variants, the dominant being the A- Iran05AFG-07 and A-Iran05BAR-08. The rate of evolution of the A-Iran05 lineage was 1.12 × 10-2 s/nt/yr. This high rate is consistent with the rapid appearance of new variants of FMDV serotype A. The A22/Iraq FMDV vaccine is antigenically distinct from A-Iran05BAR-08 viruses. Mapping of the amino acid changes between the capsid proteins of the A22/Iraq vaccine strain and the A-Iran05BAR-08 viruses onto the A22/Iraq capsid structure identified candidate amino acid substitutions, exposed on the virus surface, which may explain this antigenic difference. Phylogenetic analysis of serotype Asia-1 FMDVs revealed that three genetic Groups have circulated in Pakistan within 1998-2009. These are Group-II, -VI and a Group designated Group-VII. This new Group has not been detected in Afghanistan during the reported period but viruses from Groups I and -II are in circulation there. These studies revealed that multiple subtypes of FMDV serotypes O, A and Asia-1 co-circulate in the region and that significant new variants are frequently emerging. We have also identified an interserotypic recombinant virus, with the VP2-VP3-VP1-2A coding sequences derived from a Group-VII Asia-1 virus and the remainder of the genome from a serotype A virus of the A-Iran05AFG-07 sublineage. The Asia-1 FMDVs currently circulating in Pakistan and Afghanistan are not efficiently neutralized by antisera raised against the Asia-1/Shamir vaccine strain. Thus, new Asia-1 vaccine strains may be required to block the spread of the current Asia-1 viruses.