Hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels are important regulators of cardiac pacemaking and neuronal excitability, yet their temperature dependence has not been compared systematically across mammalian isoforms and species in standardized experimental conditions. Here, we performed a standardized electrophysiological characterization of mouse, rat and human HCN isoforms using stable CHO cell lines and automated patch‐clamp recordings across multiple temperatures and pharmacological conditions. Among the four isoforms, HCN3 did not generate detectable currents when expressed as a homomeric channel in the conditions tested, suggesting that additional factors might be required for functional activity. Comparative analysis of the remaining isoforms revealed a conserved kinetic hierarchy, with HCN1 exhibiting the fastest activation kinetics, HCN2 intermediate kinetics and HCN4 the slowest, and temperature accelerated channel gating across isoforms. Canonical modulation by cAMP and ZD7288 further showed that key regulatory features of HCN channels could be quantified reproducibly in identical experimental conditions. Together, these results provide a standardized comparative framework for interpreting HCN channel behaviour across physiological temperatures and species and establish a public reference dataset for future experimental and computational studies.