The last historic Hexagenia specimen in lower Green Bay was officially recorded in 1955. Field surveys and Hexagenia viability studies were completed to determine if lower Green Bay could support Hexagenia re-ecesis and where in the bay egg stocking could best be accomplished. The invertebrate field data were compared with historical population data based on earlier published studies in the 1950s, 1970s and 1990s to determine the bay’s ecological trajectory to better understand the re-ecesis success potential of Hexagenia. No native Hexagenia were observed during this study. Deep water invertebrate diversity within the upper lower bay appears to be improving, whereas the diversity along the lower mid-bay may be deteriorating. Shallower, nearshore samples indicated a better condition with Caenis mayflies sparsely present, amphipods, isopods, gilled snails, odonates, oligochaetes, chironomids, and meiofauna present. These results suggested improved conditions shoreward versus degraded conditions deeper. Hexagenia egg viability and neonate growth indicated Hexagenia could successfully inhabit in situ Green Bay nearshore (<2 m) substrates; however, deep substrates were generally inhospitable probably due to hypoxia and unstable fluid substrates. As an outcome of the field surveys and studies of Hexagenia viability in Green Bay mud, Hexagenia stocking began in 2014 with the first adults since 1955 emerging in 2016 at several lower bay nearshore locations. Improved water quality from remediation efforts in the watershed could facilitate the return of Hexagenia to deeper water.