Governments around the world have acknowledged that urgent action is needed to conserve and restore ecological connectivity to help reverse the decline of biodiversity. In this study we tested the hypothesis that functional connectivity for multiple species can be estimated across Canada using a single, upstream connectivity model. We developed a movement cost layer with cost values assigned using expert opinion to anthropogenic land cover features and natural features based on their known and assumed effects on the movement of terrestrial, non-volant fauna. We used Circuitscape to conduct an omnidirectional connectivity analysis for terrestrial landscapes, in which the potential contribution of all landscape elements to connectivity were considered and where source and destination nodes were independent of land tenure. Our resulting map of mean current density provided a seamless estimate of movement probability at a 300 m resolution across Canada. We tested predictions in our map using a variety of independently collected wildlife data. We found that GPS data for individual caribou, wolves, moose, and elk that traveled longer distances in western Canada were all significantly correlated with areas of high current densities. The frequency of moose roadkill in New Brunswick was also positively associated with current density, but our map was not able to predict areas of high road mortality for herpetofauna in southern Ontario. The results demonstrate that an upstream modelling approach can be used to characterize functional connectivity for multiple species across a large study area. Our national connectivity map can help governments in Canada prioritize land management decisions to conserve and restore connectivity at both national and regional scales.