In this study, after determining history of origin entering white leg shrimp of broodstocks at hatcheries of country, finally, two population of Molokaei and High health were considered as shrimp broodstocks of specific disease free (SPF) zero generation. After inbreeding and cross breeding among adult males and females of two population above, three storage different were produced in the first generation: H♂ × M♀, M♂ × H♀ and H♀ × H♂. The second generation were produced from selective breeding of M♂ × H♀. The aim of this study was to determine genetic differences between different generations of specific pathogenic free shrimp by evaluation of 16S rRNA region mitochondrial of different generations. Result of this study showed that of 486 sites have been identified 484 sites were conserved. Also, monomorphic sites ranged between 482 - 486 sites and was consists 2 polymorphic sites and 2 transitional sites. The number haplotypes, haplotypes diversity, nucleotide diversity revealed in this region were 2, 0.356±0.159 and 0.00147, respectively. Due to the high genetic identify and reduce of genetic distance of shrimp between different generations of SPF shrimp, genetic differentiation and gene flow (Nm) between them were -.142 and -2.00, respectively. This rates clearly shows a low level of polymorphism in this region of mitochondrial of different generation of SPF. This may be occurred because of small effective populations and genetic drift. Thus, offspring had inherited mitochondrial genome from your mother, therefore, it is expected that rate of variation in this genome is influenced by several factors, including genetic drift, due to unavailability of new-broodstock from different areas. It was observed that due to high conservation sites in 16S rRNA region of mitochondrial genome, nothing genetic differentiation was observed among different generations of SPF
