This study investigates the internal anatomy and petrogenesis of the Tuolumne Intrusive Suite (TIS), which comprises metaluminous, high-potassium, calc-alkaline granitoids typical of the Sierra Nevada batholith. Although the TIS has often been cited as an example of a large magma chamber that cooled and fractionated from the margins inward, its geochemistry is inconsistent with closed-system fractionation. Most major elements are highly correlated with SiO₂, but the scattered nature of trace elements and variations of initial Sr and Nd isotopic ratios indicate that fractional crystallization is not the predominant process responsible for its chemical evolution. Isotopic data suggest mixing between melts of mantle-like rocks and a granitic melt similar in composition to the highest-silica TIS unit. Monte Carlo models of magma mixing confirm that such processes can reproduce the observed variations in major elements, trace elements and isotopic ratios. Thermobarometry suggests emplacement at depths near 6 km and crystallization temperatures ranging from 660 to 750 °C. Feldspars, hornblende, biotite and magnetite exhibit evidence of extensive low-temperature subsolidus exsolution. The TIS as a whole trends toward more evolved isotopic compositions and younger U–Pb zircon ages passing inward. This pattern indicates a general increase in the proportion of felsic, crustally derived melt in the mixing process, which may have resulted from net accumulation of heat added to the lower crust by intrusion of mantle-derived mafic magma. However, the bulk geochemical and isotopic compositions of the equigranular Half Dome Granodiorite, the porphyritic Half Dome Granodiorite and the Cathedral Peak Granodiorite overlap one another and the contacts between them are commonly gradational. We interpret these map units to represent a single petrological continuum rather than distinct intrusive phases. The textural differences that define the units probably reflect thermal evolution of the system rather than distinct intrusive events.