Cucumber mosaic virus (CMV) comprises numerous isolates with various levels of in-host diversity. Subgroup-distinctive features of the Fny and LS strains provided us with a platform to genetically map the viral control elements for genetic variation in planta. We found that both RNAs 1 and 2 controlled levels of genetic diversity, and further fine mapping revealed that the control elements of mutation frequency reside within the first 596 amino acids (aa) of RNA 1. The 2a/2b overlapping region of the 2a protein also contributed to control of viral genetic variation. Furthermore, the 3' nontranslated region (NTR) of RNA 3 constituted a hot spot of polymorphism, where the majority of fixed mutations found in the population were clustered. The 2b gene of CMV, a viral suppressor of gene silencing, controls the abundance of the fixed mutants in the viral population via a host-dependent mechanism.