Tributary and mainstem connections represent important links for the movement of fish and other biota throughout river networks. We investigated the timing, frequency and environmental conditions associated with movements by adult golden perch (Macquaria ambigua) between the mainstem of the mid-Murray River and a tributary, the Goulburn River, in south-eastern Australia, using acoustic telemetry over four years (2007-2011). Fish were tagged and released in autumn 2007-2009 in the mid-Murray (n = 42) and lower Goulburn (n = 37) rivers within 3-6 km of the mid-Murray-lower Goulburn junction. 38% of tagged fish undertook mainstem-tributary movements, characterised mostly by temporary occupation followed by return of fish to the original capture river. Approximately 10% of tagged fish exhibited longer-term shifts between the mainstem and tributary. Movement of fish from the tributary into the mainstem occurred primarily during the spawning season and in some years coincided with the presence of golden perch eggs/larvae in drift samples in the mainstem. Many of the tributary-to-mainstem movements occurred during or soon after changes in flow. The movements of fish from the mainstem into the tributary were irregular and did not appear to be associated with spawning. The findings show that golden perch moved freely across the mainstem-tributary interface. This demonstrates the need to consider the spatial, behavioural and demographic interdependencies of aquatic fauna across geographic management units such as rivers.