Understanding the reasons and cues for migration is crucial for developing effective conservation and management strategies of diadromous fishes. Spawning and movement patterns of the threatened diadromous Australian grayling (Prototroctes maraena) were investigated in the Bunyip River, Victoria, using drift sampling (2008–2011) and acoustic telemetry (2009–2010) during the autumn–winter spawning period of each year. Fifty-five adult fish (2009: n = 21; 2010: n = 34) were tagged and released in February ~15–30 km upstream of the Bunyip River estuary. Thirteen fish (2009: n = 7; 2010: n = 6) undertook rapid downstream migrations from March to April to reaches immediately upstream of the estuary. Drifting eggs were detected at multiple sites between April and July; however, the majority (78.8%) were collected in the lower reaches within ~0.5 km of the estuary in early–mid-May. Tagged adult fish arrived in this area 1–4 weeks before eggs were detected and usually moved back upstream within 2 weeks following the peak egg abundance. Downstream migration and peak egg abundance were associated with increased river flows. Although the proportion of fish that undertook migrations was low, low rates of tag retention in this species likely account for the failure to detect migration by many of the tagged individuals.