We used stable hydrogen isotopes (δD) to identify the breeding locations of Wilson’s Warbler (Wilsonia pusilla) migrating through five sites spanning a cross-section of the species’ southwestern migration route during the springs of 2003 and 2004. Determining the temporal and spatial patterns of migration and degree of population segregation during migration is critical to understanding long-term population trends of migrant birds. At all five migration sites, we found a significant negative relationship between the date Wilson’s Warblers passed through the sampling station and δD values of their feathers. These data were consistent with a pattern of “leap-frog” migration, in which individuals that bred the previous season at southern latitudes migrated through migration stations earlier than individuals that had previously bred at more northern latitudes. We documented that this pattern was consistent across sites and in multiple years. This finding corroborates previous research conducted on Wilson’s Warbler during the fall migration. In addition, mean δD values became more negative across sampling stations from west to east, with the mean δD values at each station corresponding to different geographic regions of the Wilson’s Warblers’ western breeding range. These data indicate that Wilson’s Warblers passing through each station represented a specific regional subset of the entire Wilson’s Warbler western breeding range. As a result, habitat alterations at specific areas across the east-west expanse of the bird’s migratory route in the southwestern United States could differentially affect Wilson’s Warblers at different breeding areas. This migration information is critical for management of Neotropical migrants, especially in light of the rapid changes presently occurring over the southwestern landscape.