Differences in sequence homology between human (h), mouse (m), and rat (r) histamine H<sub>4</sub> receptors (H<sub>4</sub>R) cause discrepancies regarding affinities, potencies, and/or efficacies of ligands and therefore compromise translational animal models and the applicability of radioligands. Aiming at a radioligand enabling robust and comparative binding studies at the h/m/rH<sub>4</sub>Rs, 2,4-diaminopyrimidines were synthesized and pharmacologically investigated. The most notable compounds identified were two (partial) agonists with comparable potencies at the h/m/rH<sub>4</sub>Rs: UR-DEBa148 (<i>N</i>-neopentyl-4-(1,4,6,7-tetrahydro-5<i>H</i>-imidazo[4,5-<i>c</i>]pyridin-5-yl)pyrimidin-2-amine bis(2,2,2-trifluoroacetate), <b>43</b>), the most potent [pEC<sub>50</sub> (reporter gene assay) = 9.9/9.6/10.3] compound in the series being slightly G-protein biased and UR-DEBa176 [(<i>R</i>)-4-[3-(dimethylamino)pyrrolidin-1-yl]-<i>N</i>-neopentylpyrimidin-2-amine bis(2,2,2-trifluoroacetate), <b>46</b>, pEC<sub>50</sub> (reporter gene assay) = 8.7/9.0/9.2], a potential "cold" form of a tritiated H<sub>4</sub>R ligand. After radiolabeling, binding studies with [<sup>3</sup>H]UR-DEBa176 ([<sup>3</sup>H]<b>46</b>) at the h/m/rH<sub>4</sub>Rs revealed comparable <i>K</i><sub>d</sub> values (41/17/22 nM), low nonspecific binding (11-17%, ∼<i>K</i><sub>d</sub>), and fast associations/dissociations (25-30 min) and disclosed [<sup>3</sup>H]UR-DEBa176 as useful molecular tool to determine h/m/rH<sub>4</sub>R binding affinities for H<sub>4</sub>R ligands.