Filling of the enlarged Cotter Reservoir commencing in mid-2013 saw a cessation in recruitment for the resident population of endangered Macquarie perch. Recruitment failure was attributed to the headwaters abutting natural instream barriers as it filled, preventing access to spawning habitat. A full reservoir 2016 resulted in inundation of barriers and successful recruitment, though characteristics and location of spawning habitat remain unknown in this catchment. This study aimed to determine the location and characterise the microhabitat features of Macquarie perch spawning habitat in the lower Cotter River upstream of Cotter Reservoir. Twelve riffles from three reaches in the lower Cotter River were sampled for Macquarie perch eggs fortnightly from October – November 2017. Eggs sampling and microhabitat characteristics of stratified quadrats were measured for each riffle. Macquarie perch eggs were detected from one reach (the reach immediately upstream of Cotter Reservoir) and on three of the four riffles within this reach. There was no detectable difference in microhabitat characteristics between riffles containing eggs and those that did not. Failure to detect Macquarie perch eggs in the upstream-most two reaches may be because of sampling intensity within riffle or spawning occurring in adjacent riffles that were not sampled.