In order to estimate the effective population size (Ne) in Iranian water buffalo blood and hair samples of 407 individual from Azari (N=260), Khuzestani (N=120) and Mazandrani (N=27) buffalo populations were gathered. After DNA extaraction, the samples were genotyped using Axiom® Buffalo Genotyping 90K Array. The Ne was estimated from 700 to 4 generations ago and also for the present generation by linkage disequiblirum data and based on heterozygote-excess method using NeEstimator (V2), respectively. Estimated Ne for Azari, Khuzestani and Mazandarani were calculated 1530, 1375 and 1141, respectively, for 700 generations ago. Ne for the present generation in Azeri, Khuzestani and Mazandarani were estimated 447, 226 and 35, respectively. The Ne for Azeri and Khuzestani were relatively high and these two populations were not endanger to extinction, but their Ne has been declined in the resent generations massively and it is necessary to care about the maintenance of Ne and relatively high diversity for these populations. However, the Mazandarani population is endangered because of low Ne and so it is necessary to carefully monitor their effective population size, improve the profitability of production and planning a suitable mating scheme to control inbreeding and genetically conserve this population.