Determining how species move across complex and fragmented landscapes and interact with human-made barriers is a major research focus in conservation. Studies estimating functional connectivity from movement, dispersal, or gene flow usually rely on a single study period, and rarely consider variation over time. We contrasted genetic structure and gene flow across barriers for a metapopulation of desert bighorn sheep (Ovis canadensis nelsoni) using genotypes collected 2000–2003 and 2013–2015. Based on the recently observed but unexpected spread of a respiratory pathogen across an interstate highway previously identified as a barrier to gene flow, we hypothesized that bighorn sheep changed how they interacted with that barrier, and that shifts in metapopulation structure influenced gene flow, genetic diversity, and connectivity. Population assignment tests, genetic structure, and genetic recapture demonstrated that bighorn sheep crossed the interstate highway in at least one location in 2013-2015, sharply reducing genetic structure between two populations, but supported conclusions of an earlier study that such crossings were very infrequent or unknown in 2000-2003. A recently expanded population established new links and caused decreases in genetic structure among multiple populations. Genetic diversity showed only slight increases in populations linked by new connections. Genetic structure and assignments revealed other previously undetected changes in movements and distribution, but much was consistent. Thus, we observed changes in both structural and functional connectivity over just two generations, but only in specific locations. Movement patterns of species should be revisited periodically to enable informed management, particularly in dynamic and fragmented systems.