Maintaining landscape connectivity where habitat linkages or animal migrations intersect roads requires some form of mitigation to increase permeability. Wildlife crossing structures are now being designed and incorporated into numerous road construction projects to mitigate the effects of habitat fragmentation. For them to be functional they must promote immigration and population viability. There has been a limited amount of research and information on what constitutes effective structural designs. One reason for the lack of information is because few mitigation programs implemented monitoring programs with sufficient experimental design into pre- and post-construction. Thus, results obtained from most studies remain observational at best. Furthermore, studies that did collect data in more robust manners generally failed to address the need for wildlife habituation to such large-scale landscape change. Such habituation periods can take several years depending on the species as they experience, learn and adjust their own behaviours to the wildlife structures. Also, the brief monitoring periods frequently incorporated are simply insufficient to draw on reliable conclusions. Earlier studies focused primarily on single-species crossing structure relationships, paying limited attention to ecosystem-level phenomena. The results of single species monitoring programs may fail to recognize the barrier effects imposed on other non-target species. Thus, systems can be severely compromised if land managers and transportation planners rely on simple extrapolation species. In a previous analysis of wildlife underpasses in Banff National Park (BNP), Canada, we found human influence consistently ranked high as a significant factor affecting species passage. Our results suggest that the physical dimensions of the underpasses had little effect on passage because animals may have adapted to the 12-year old underpasses. As a sequel to the above study, we examined a completely new set of recently constructed underpasses and overpasses which animals had little time to become familiar with. We investigated the importance of temporal and spatial variability using data obtained from systematic, year-round monitoring of 13 newly-constructed wildlife crossing structures 34 months post-construction. Our results suggest that structural attributes best correlated to performance indices for both large predator and prey species, while landscape and human-related factors were of secondary importance. These findings underscore the importance of integrating temporal and spatial variability as a priori when addressing wildlife crossing structure efficacy, and the fact that species respond differently to crossing structure features. Thus mitigation planning in a multiple-species ecosystem is likely to be a challenging process. The results from this work suggest that mitigation strategies need to be proactive at the site and landscape level to ensure that crossing structures remain functional over time, including human use management. Continuous long-term monitoring of crossing structures will be key to ascertaining the strengths and weaknesses of design characteristics for a multi-species assemblage