Persistent plant viruses circulate between host plants and vector insects, possibly leading to the genetic divergence in viral populations. We analyzed the single nucleotide polymorphisms (SNPs) of Rice stripe virus (RSV) when it incubated in the small brown planthopper and rice. Two SNPs, which lead to nonsynonymous substitutions in the disease-specific protein (SP) of RSV, produced three genotypes, i.e., GG, AA and GA. The GG type mainly existed in the early infection period of RSV in the planthoppers and was gradually substituted by the other two genotypes during viral transmission. The two SNPs did not affect the interactions of SP with rice PsbP or with RSV coat protein. The GG genotype of SP induced stronger immune responses than those of the other two genotypes in the pattern recognition molecule and immune-responsive effector pathways. These findings demonstrated the population variations of RSV during the circulation between the vector insect and host plant.