Potassium currents are essential for physiological plasticity of cardiac function in vertebrates. In cardiac myocytes, the inward rectifier K⁺ current (I_K1) sets the resting membrane potential and contributes to the phase 3 repolarization of action potential. In trout (Oncorhynchus mykiss) heart, I_K1 is modified by temperature acclimation. The aim of the present study was to examine the molecular basis of cardiac I_K1 in trout acclimated to cold (4°C, c.a.) and warm (18°C, w.a.) temperature. RT-PCR was used to clone Kir2.1 and Kir2.2 channels, qPCR to determine Kir2 transcripts, and single-channel and whole-cell patch-clamp to examine I_K1 properties. Electrophysiological characteristics of the I_K1 were changed by temperature acclimation. Ba²⁺ sensitivity of the I_K1 was nearly ten times higher in w.a. (IC₅₀, 0.18 ± 0.42 mM) than in c.a. (IC₅₀, 1.17 ± 0.44 mM) trout, and the crossover point of the outward I_K1 at 5.4 mM and 10.8 mM extracellular K⁺ occurred at more negative voltage in w.a. (-45.88 ± 1.66 mV) than in c.a. (-39.75 ± 1.77 mV) trout. Single-channel conductance was not different, but opening kinetics was faster in c.a. than w.a. trout. Despite the functional differences, the expression of Kir2.1 and Kir2.2 transcripts was not altered by thermal acclimation. It is concluded that temperature-induced changes in trout cardiac I_K1 are not due to different Kir2.1-2 channel expression, but might be due to different heteromeric composition of Kir2 channels or differential regulation of channels by intracellular polyamines and membrane cholesterol.