Subsurface impacts associated with Marine Heatwaves (MHWs) in estuaries are not well understood, largely due to data scarcity. Using over three decades (1986–2021) of observations from several monitoring programs, this study investigates subsurface temperature and dissolved oxygen (DO) anomalies associated with surface MHWs in the Chesapeake Bay (CB). Seasonal variability in temperature anomalies followed a simple 1‐D response to surface heating with downward heat transport and diffusion controlled by seasonally variable stratification and mixing. Two distinct temperature regimes were found: a thermally stratified spring‐summer regime, when positive temperature anomalies were confined to the surface mixed layer (SML); and a homogeneous fall‐winter regime. Additionally, surface (subsurface) temperatures were elevated for months (days to weeks) before and after MHWs, indicating individual events were shorter than the timescales of elevated temperatures. A simple 1‐D SML heat budget identified air‐estuary heat flux changes as the leading driver of MHW onsets and declines, with latent heat flux being the dominant term. DO anomaly patterns were more complex, with considerable along‐channel gradients. Notable DO decreases (1–4 mg L−1) primarily occurred in the winter/spring below the SML, and the hypoxic zone expanded from spring through fall. Only a small fraction of these DO anomalies could be attributed to MHW temperature‐induced solubility changes, demonstrating that other physical and/or biogeochemical processes dominate changes in DO during these events. In CB, concurrent low DO and persistent high temperatures could compound the impacts of MHWs on this valuable ecosystem, with MHW event impacts likely to be exacerbated by climate change.