Aims It is unclear how changing atmospheric conditions, including rising carbon dioxide concentration, influence interactions between above and below-ground systems and if intraspecific variation exists in this response. Methods We assessed interactive effects of atmospheric CO2 concentration, above-ground herbivory, and plant genotype on root traits and mycorrhizal associations. Plants from five families of Asclepias syriaca, a perennial forb, were grown under ambient and elevated atmospheric CO2 concentrations. Foliar herbivory by either lepidopteran caterpillars or phloem-feeding aphids was imposed. Mycorrhizal colonization, below-ground biomass, root biomass, and secondary defensive chemistry in roots were quantified. Results We observed substantial genetic variation among A. syriaca families in their mycorrhizal colonization levels in response to elevated CO2 and herbivory treatments. Elevated CO2 treatment increased root biomass in all genetic families, whereas foliar herbivory tended to decrease root biomass. Root cardenolide concentration and composition varied greatly among plant families, and elevated CO2 treatment increased root cardenolides in two of the five plant families. Moreover, herbivores differentially affected the composition of cardenolides expressed below ground. Conclusions Increased atmospheric CO2 has the potential to influence interactions among plants, herbivores and mycorrhizal fungi and intraspecific variation suggests that such interactions can evolve.