It is about 50 years that FMD affected the ruminants of Isfahan. Last outbreaks of FMD were happened at 2005 even vaccinated animals, so in current work using RT-PCR, sequencing and regression "r" values, the isolated strains in Isfahan were identified. The aim of this study was molecular epidemiology of FMDV in Isfahan province as the central part of Iran in 2006-2009. According to the result , a highly pathogen A05 strain was isolated from west (Najafabad city) about 2 months after the entrance of this virus to Iran through the west and north west margins toward central part and then distributed around 10 cities of Isfahan province. Here it is obvious that the A05 strain of Isfahan just showed 1% difference with A05IR (vaccine strain), in which for A22 were 65 %. Also based on the alignment of 600 bp of 3΄ end of the VP1 sequences of isolated type O comparing with representative of type O Shabestar vaccine strain and the other provinces of Iran, the Isfahan O isolate was 3% distinct from O shabestar vaccine strain. In a random "r" value detection of west isolate strain (A /Najafabad/Isfahan/Iran/ 05) against A87IR were 0.35 and against A05IR were 0.73; For O strain, randomly "r" value of center isolate (O/Isfahan/Isfahan/Iran) obtained against Iranian O vaccine strain (O Shabestar) were 0.76 and with O 967 (Panasia) were 0.88. Regarding to the conclusion, the FMD lived vaccine for Isfahan was improved with A05/Ir FMDV by Razi Vaccine and Serum Research Institute (RVSRI).