Heart rate of the freshwater turtle (Trachemys scripta) is markedly depressed with anoxia exposure and arises in part from a resetting of intrinsic heart rate. Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels (“pacemaker” channels) constitute an important component in the control of cardiac rhythmicity. HCN channels are tetramers, and four HCN channel subunit isoforms exist, which are encoded for by four genes (HCN1-4). The four isoforms can form homotetramers and heterotetramers, resulting in a number of HCN channels with distinct biophysical properties. Using real-time RT-PCR, we examined the effects of anoxia and subsequent reoxygenation on the expression of HCN1-4 mRNA in the cardiac chambers (sinus venosus, right atrium, left atrium and ventricle) of 21°C- and 5°C-acclimated turtles. With 24 h of anoxia at 21°C, the relative abundance of HCN2 increased in all cardiac chambers, and following 24 h reoxygenation, HCN2 expression returned to control levels. In contrast, at 5°C, few and only minor changes in HCN channel subunit expression occurred with anoxia (14 d) and reoxygenation (13 d). However, compared to 21°C normoxic turtles, cardiac chambers of 5°C normoxic turtles exhibited a pronounced increase in the relative abundance of HCN2. Combined, the findings suggest that modified HCN expression with cold acclimation may prime the turtle cardiac muscle for the approaching anoxic winter, and that cardiac anoxia survival at 21°C may be aided by the circumvention of this priming and through rapid induction of similar changes in HCN expression. The altered HCN expression may serve to facilitate a slowed heart rate during anoxia by fine-tuning the sensitivity of HCN channels to the changing levels of intracellular and extracellular modulators of HCN activity that occurs with anoxia exposure. Indeed, the turtle HCN isoforms show high sequence similarity to their mammalian counterparts, notably at the location of key regulatory motifs.