Biodiversity among domesticated animals in developing countries is enormous. However, due to introduction of superior animal breeds with excellent performance, the native animal resources with good adaptability but lower productivity are in great danger. The list of extinct local breeds and deteriorated remaining ones are becoming longer every year. Erosion of genetic diversity in a breed may cause increase in the rate of inbreeding and genetic abnormalities thereby decrease in animal performance, particularly for reproductive traits. These will virtually reduce the global gene pool for future development and can be considered as a serious threat for universal food security. Therefore the urgency and need to conservation of genetic resources in animal biodiversity is clear particularly for those in the developing countries (Hall, et al. 1995). Buffalo play an important role in animal production in some province of Iran. Present study follow to evaluate the genetic diversity and existing relationship among three Iranian native buffalo populations by using microsatellite markers.