The Kv1.3 voltage-gated K+ channel is primarily expressed in immune cells and the CNS. Functional heterotetrameric channels composed of different Kv1 subunit combinations are common in the brain. Kv1.3 subunits can associate with Kv1.1–Kv1.6 subunits to form heterotetrameric channels. Potent inhibitors of homotetrameric Kv1.3 include native or modified peptide toxins such as ShK-186, HsTX1[R14A], and Vm24, which exhibit hundred- to several thousand-fold selectivity for Kv1.3 over other physiologically important ion channels. However, their activity against asymmetric heteromeric channels expressed in different cell types remains largely unexplored. Therefore, limited information is available on the activity of Kv1.3-selective peptide toxins against heterotetrameric channels. Targeting heteromeric channels containing various numbers of Kv1.3 subunits by these toxins may shed light on potential side effects during systemic application. To systematically study the pharmacology of Kv1.3 heterotetramers, we generated concatenated dimers and tetramers by tandem-linking Kv1.3 α-subunits with other Kv1.x subunits. All constructs exhibited robust expression in mammalian cells. Fluorescence-detection size-exclusion chromatography (FSEC) analysis of solubilised membrane proteins suggested that the dimer constructs assembled with the expected stoichiometry. Electrophysiological analysis demonstrated that tandem linking did not alter the biophysical or pharmacological properties of homotetrameric channels assembled from Kv1.3-Kv1.3 dimers. In contrast, heterotetrameric Kv1.3-Kv1.x channels displayed intermediate K+ current inactivation kinetics, and their affinity for tetraethylammonium (TEA) followed the expected dependence on the ratio of TEA-sensitive and TEA-insensitive Kv1 subunits, confirming heteromeric channel assembly. All three toxins exhibited a wide range of affinities for Kv1 heterotetrameric channels. Vm24 showed >100-fold lower affinity for all Kv1.3 heteromeric channels than for Kv1.3 homomers, except Kv1.3-Kv1.2 (14-fold lower affinity; IC50 = 64 pM). HsTX1[R14A] exhibited >40-fold lower affinity for all Kv1.3 heteromeric channels than for Kv1.3 homotetramers except Kv1.3-Kv1.1 (15-fold lower affinity; IC50 = 64 pM). ShK-186 maintained high affinity for Kv1.3-Kv1.1, Kv1.3-Kv1.2, and Kv1.3-Kv1.6 heterotetramers (IC50 = 7–25 pM), comparable to the Kv1.3 homomeric channel, but exhibited high selectivity for Kv1.3 over Kv1.3-Kv1.4 and Kv1.3-Kv1.5 heterotetramers. These differences in potency likely reflect unique interactions between toxins and asymmetric heteromeric channels. These findings provide a framework for the rational design of peptide inhibitors that selectively target Kv1.3 homomeric channels over Kv1 heteromeric channels and for developing Kv1.3-Kv1.5 heteromer-specific inhibitors to target microglial cells in the brain.