Binding to various tissues and species is frequently assessed in drug discovery and development to support safety and efficacy studies. To reduce time, cost, and labor requirements for binding experiments, we conducted a large comparison study to evaluate the correlation of fraction unbound (f u ) across 7 tissues of 5 species, including white adipose, brain, heart, kidney, liver, lung, and skeletal muscle of mouse, rat, dog, monkey, and human. The results showed that there were no significant species differences of f u for tissue binding, and a single-species (e.g., rat) tissue f u can be used as a surrogate for binding in other species. Cross-tissue comparison indicated that brain, heart, liver, and muscle had quite similar f u values; rat liver binding can be used as a surrogate for binding of the other 3 tissues without any scaling factors. Binding to adipose, kidney, and lung can also be estimated with rat liver f u with scaling factors. This study suggests that a single tissue of a single species (e.g., rat liver) is a good predictor for f u of other tissues of various species with or without scaling factors. Molecular size, lipophilicity, pKa, and topological polar surface area are important physiochemical properties influencing tissue f u .