Connectivity plays a key role in biodiversity conservation as it sustains ecological processes important for the maintenance of populations such as migrations. Connectivity is especially relevant for species that rely on different realms during their life cycle or use different realms daily or seasonally (multi-realm species). However, effort to address conservation across multiple realms have focused on identifying priority areas for conservation in a single realm (mostly marine) accounting for threats propagating from other realms or single species needs. Here, we demonstrate how to identify priority areas for conservation across three different realms (freshwater-terrestrial, estuary and marine) for multiple species, including some multi-realm species, that inhabit or move across the three realms. We use the whole Tagus River Basin, its estuary and nearby marine area as a case study. We compared the allocation of priority areas and spatial connectivity achieved under three scenarios: no-connectivity, within-realm and cross-realm connectivity scenarios. There were some differences in the spatial allocation of priority areas across scenarios. The most remarkable difference laid on the connectivity achieved under each scenario, which experienced a 3-fold increase when considering connectivity across realms, compared to solutions that considered only connectivity within each realm independently. This improvement in connectivity was especially marked for some of the species that occur across the three realms. There were, however, trade-offs between this improvement in connectivity: i) an increase in the number of planning units selected, especially in the estuary, the realm that links the other two; and ii) a decline in connectivity achieved within the freshwater—terrestrial and marine realms. Synthesis and applications: Addressing connectivity across realms deserves especial attention when planning for conservation of multi-realm species to ensure adequacy of conservation recommendations to respond to the needs of these species. Given the potential trade-offs between enhanced cross-realm connectivity and total area needed or internal within-realm connectivity, consideration of cross-realm connectivity must be cautiously evaluated and integrated in multi-realm conservation plans.