Genetic variability was studied on Mazandaran and Isfahan native chicken populations based on twenty microsatellite markers (MCWOOI4, MCW0081, MCWOI83, MCW0067, MCWOI04, MCWOI23, MCW0330, MCWOI65, MCW0069, MCW0020, MCW0222, LEI0094, MCW0295, MCW0034, MCW0216, ADL0268, ADLO 112, ADL0278 and LEIO 166). Blood samples of 90 and 150 individuals were randomly collected from Mazandaran and Isfahan native chicken populations, respectively. Genomic DNA was isolated using optimized and modified salting-out procedure. The number of ALLELEs varied from I to 6. One locus (MCW0216) in Mazandaran population and three loci (MCW0216, MCW67 and MCW222) in Isfahan population were monomorphic. The other loci were shown appropriate polymorph isms. All microsatellite loci were in Hardy- Weinberg equilibrium except for MCW222 and MCWI65 loci in Mazandaran population (p<0.005). The observed heterozygosity ranged from 0.7437 (LEI0094) to 0.2472 (MCWI65) in Isfahan and Mazandaran populations, respectively. The least EFFECTIVE ALLELE value belongs to locus MCW216, in both of the populations, and the highest belongs to LEI009410cus in Isfahan chickens. The large stand the lowest PIC values were observed in MCWO 104 locus in Isfahan and in MCW 165 locus in Mazandaran populations, respectively. Loci with two ALLELEs were showed the least amount of Shannon index in both populations. Loci with more ALLELEs had more Shannon index. Generally, polymorphisms in the studied experimental populations were relatively low.