Equine infectious anemia virus (EIAV) exhibits a high rate of genetic variation in vivo and results in a clinically variable disease in infected horses. Previous studies identified distinct subpopulations within the Rev quasispecies of an experimentally infected pony, pony 524. The subpopulations showed significantly different phenotypes, as measured by in vitro assays, and fluctuated in dominance in a manner coincident with clinical stage of disease. This study further characterizes the genotypic and phenotypic variation within and between the previously identified EIAV Rev subpopulations in pony 524, and characterizes the EIAV Rev quasispecies of another EIAV-infected pony, pony 625. Within pony 524, the Rev protein was highly conserved and only ten amino acid mutations were found at high frequency within the entire Rev quasispecies. Nine of these amino acid mutations were capable of significantly altering Rev activity, either as a single mutation in the context of the founder variant, R1, or in the context of cumulatively fixed mutations. Phylogenetic analysis indicated nine of these ten mutations were fixed into the high Rev activity subpopulation over the course of disease. The fixation of the amino acid mutations, however, did not confer an increase in Rev activity over time; rather, it maintained the high Rev phenotype of this subpopulation. Greater differences in Rev phenotype were observed between subpopulations, rather than within subpopulations of EIAV Rev as they evolved throughout disease. The greatest difference in Rev phenotype was observed between the two subpopulations of EIAV Rev early in disease, and both subpopulations maintained their respective Rev phenotype throughout disease. The characterization of the Rev quasispecies in pony 625 identified two coexisting subpopulations that differed in phenotype, as observed in pony 524. This provides further evidence that viral quasispecies are composed of coexisting, independently evolving subpopulations that differ in phenotype. The persistence of minor, less fit subpopulations would allow the viral quasispecies to quickly adapt to changes within its environment through the expansion of minor subpopulations. Further study of multiple coexisting subpopulations will provide additional insight into the nature of quasispecies evolution and lentiviral persistence.