Rainbow trout ( Oncorhynchus mykiss ) of Salmonidae family is one of the most important fish farmed in Iran. Considering the importance of genetic variation and population structure for management and broodstock selection for replication competent in the farms, the genetic diversity of rainbow trout was investiged using molecular techniques. In this study 30 specimens of rainbow trout were collected from each of four populations (Iranian, American, Spanish and French). For molecular study, DNA was extracted using ammonium acetate method from fin clips and/or part of bodies. The DNA quality was examined by electrophoresis on 1% agaros gel and painted with ethidium bromide. PCR amplification was performed using four pairs of microsatellite primers (OMY77, OMY325, OMM1329 and OMM1332) at annealing temperatures of 58.5-62.5. PCR products were electrophoresed on 12% achrylamid gel and stained with silver nitrate. Calculations was done using GenAlex.6 software. All loci were polymorphic. Number of observed alleles were in the range of 8-16. Average number of observed alleles in Iranian, American, Spanish and French populations were 10.75 , 11 , 11.5 and 10 to respectively. The number of effective alleles were in the range of 5.40-11.53. Average number of effective alleles in Iranian, American, Spanish and French populations were 7.43, 8.03 , 8.65 and 7.19 respectively. Allele sizes at OMY77, OMY325, OMM1329 and OMM1332 loci were in the range of 102-178, 100-150, 122-198, 172-204 respectively. The means of observed heterozygosities in Iranian, American, Spanish and French populations were 0.591, 0.591, 0.633 and 0.566 to respectively. The mean of expected heterozygosity for Iranian , American, Spanish and French populations were 0.861, 0.868, 0.879 and 0.854 respectively. Results showed deviation from Hardy-Weinberg equilibrium at fourteen out of sixteen studied loci. Increase of observed heterozygosity towards expected heterozygosity at OMY325 loci in Iranian, OMY325 in American, OMY325 in Spanish and French populations, low (7%) genetic segregation between population, high (93%) genetic diversity within population and high (16.07) gene flow were observed. Genetic identity and genetic distance between all population were in the range of 0.727- 0.847 and 0.166-0.319 respectively. A tree diagram of the four populations based on Nei (1972, 1978) genetic distance and UPGMA method was performed. Based on the results, American, Spanish and French populations were put in a same branch while Iranian population was placed in separate branch. The F ST was measured as 0.023 and F IS in Iranian, American, Spanish and French populations were 0.329, 0.334, 0.295 and 0.343 respectively. As regards to separation of Iranian population from other populations, it can be said that for implementation of conflux program of two populations for hybridization, the best choice is conflux Iranian population with one of the other populations. The genetic diversity value of Spanish population was more than others. For better understanding of genetic structure of cultivated rainbow trout, as one of the most important cultivated species in Iran, study on all cultivated stocks using higher number of microsatellite loci or other molecular markers is highly recommend. Keyword: Rainbow trout; Oncorhynchus myki Microsatellite ; Genetic diversity; Genetic variation.