Neuropeptide Y (NPY) receptors (YRs) play a key regulatory role in essential human physiological processes such as bone metabolism, feeding, anxiolysis, and pain modulation, making them potential therapeutic targets. Nonetheless, radiochemical ligand binding studies needed for the development of therapeutics actings at YRs, have been conducted inconsistently in the presence or absence of sodium, the latter representing a nonphysiological-like condition that may lead to inaccurate interpretations. As such, this study aimed to provide an in-depth overview of the binding affinities of endogenous YR agonists, namely NPY, peptide YY (PYY), and pancreatic polypeptide (PP), to the various YR subtypes, Y1R, Y2R, Y4R, and Y5R, in the presence and absence of sodium using recently developed tritium-labeled radioligands. Additionally, YR subtype-selective antagonists, BIBP3226 (Y1), JNJ31020028 and BIIE0246 (Y2), UR-MK188 (Y4), and CGP71683A (Y5), were studied. Significant differences in YR binding of the agonists, observed for Y1R, Y4R and Y5R binding, were indicated by higher binding affinities in the absence of sodium. The effect was most pronounced for Y1R binding of PP (ca. 100-fold difference in binding affinity). While Y2R binding of the Y2R antagonist BIIE0246 and Y4R binding of the putative Y4R antagonist UR-MK188 were not significantly influenced, a significantly decreased binding affinity was observed for the antagonists BIBP3226 (Y1R) and CGP71683 (Y5R) for binding under sodium-free conditions compared to the sodium-containing buffer. Overall, the determined YR binding affinities were largely consistent with literature data. Findings from this study confirmed sodium’s allosteric modulation on YR agonist binding, although this modulatory effect was shown to be dependent on the type of ligand bound.