<p>Viral host tropism defines the molecular boundaries of infection and governs the adaptive processes by which pathogens cross species barriers. This review synthesizes the mechanistic and evolutionary determinants that enable viral spillover, with a focus on receptor recognition, proteolytic activation, immune evasion, and genomic plasticity as core drivers of host range expansion. By integrating structural virology, comparative genomics, and molecular immunology, we examine how adaptive mutations, recombination, and host‒virus coevolution shape molecular compatibility and facilitate interspecies transmission. We further analyzed how environmental and anthropogenic pressures, including habitat disruption, climate change, and wildlife‒livestock‒human interfaces, modulate viral evolution and increase exposure opportunities. Central to this synthesis is the proposed sequential molecular gatekeeping model of viral spillover, which outlines the stepwise molecular and ecological barriers that viruses must overcome to achieve successful cross-species emergence. This review highlights how advances in metagenomic surveillance and artificial intelligence (AI)-driven prediction of receptor binding affinity can identify high-risk viral lineages before they cause outbreaks. Framed within a One Health perspective, this work bridges molecular mechanisms, ecological dynamics, and translational preparedness, offering a conceptual framework to support hypothesis generation and risk-informed surveillance. The SMGM integrates previously fragmented molecular determinants into a structured, stage-based conceptual model rather than introducing entirely new mechanistic components.</p>