The association between perennial ryegrass (Loliumperenne L.) and its Epichloë fungal endophyte symbiont, Epichloëfestucae var. lolii, supports the persistence of ryegrass-based pastures principally by producing bioactive alkaloid compounds that deter invertebrate herbivory. The host plant genotype affects endophyte trait expression, and elucidation of the underlying genetic mechanisms would enhance understanding of the symbiosis and support improvement of inplanta endophyte performance through plant breeding. Rapid metabolite profiling and enzyme-linked immunosorbent assay were used to quantify endophyte alkaloids and mycelial mass (MM) in leaves harvested, in consecutive autumns, from an F1 mapping population hosting standard toxic endophyte. Co-aligned quantitative trait loci (QTL) on linkage groups (LG)2, LG4 and LG7 for MM and concentrations of alkaloids peramine and ergovaline confirmed host plant effects on both MM and alkaloid level and inferred the effect on alkaloids was modulated through the quantity of endophyte present in the leaf tissue. For ergovaline, host regulation independent of endophyte concentration was also indicated, by the presence of MM-independent ergovaline QTL on LG4 and LG7. Partitioning of host genetic influence between MM-dependent and MM-independent mechanisms was also observed for the alkaloid N-formylloline (NFL), in a second mapping population harbouring a tall fescue-sourced endophyte. Single-marker analysis on repeated MM and NFL measures identified marker-trait associations at nine genome locations, four affecting both NFL and MM but five influencing NFL concentration alone. Co-occurrence of QTL on LG3, LG4 and LG7 in both mapping populations is evidence for host regulatory loci effective across genetic backgrounds and independent of endophyte variant. Variation at these loci may be exploited using marker-assisted breeding to improve endophyte trait expression in different host population × endophyte combinations.