A remarkable number of SARS-CoV-2 variants and other as yet unmonitored lineages harbor amino-acid substitutions with the potential to modulate the interface between the spike receptor-binding domain (RBD) and its receptor ACE2. The naturally occurring Q498Y substitution, which is present in currently circulating SARS-CoV-2 variants, has drawn the attention of several investigations. While computational predictions and in vitro binding studies suggest that Q498Y increases the binding affinity of the spike protein for ACE2, experimental in vivo models of infection have shown that a triple mutant carrying the Q498Y replacement is fatal in mice. To accurately characterize the binding kinetics of the RBD Q498Y–ACE2 interaction, biolayer interferometry analyses were performed. A significant enhancement of the RBD–ACE2 binding affinity relative to a reference SARS-CoV-2 variant of concern carrying three simultaneous replacements was observed. In addition, the RBD Q498Y mutant bound to ACE2 was crystallized. Compared with the structure of its wild-type counterpart, the RBD Q498Y–ACE2 complex reveals the conservation of major hydrogen-bond interactions and a more populated, nonpolar set of contacts mediated by the bulky side chain of Tyr498 that collectively lead to this increase in binding affinity. In summary, these studies contribute to a deeper understanding of the impact of a relevant mutation present in currently circulating SARS-CoV-2 variants which might lead to stronger host–pathogen interactions.