A new set of 18 microsatellite loci was developed for the threatened Australian freshwater fish southern purple-spotted gudgeon Mogurnda adspersa (Eleotridae) using a next generation sequencing approach. A total of 84 fish from two populations (including one rescued into captivity) were successfully genotyped at all markers using a multiplex approach. As expected for threatened species, we observed relatively low genetic variation across most loci (average allelic diversity = 5.4; average heterozygosity = 0.380). No evidence for linkage disequilibrium was detected and all loci were in Hardy–Weinberg equilibrium. This new set of microsatellite markers will benefit substantially the ongoing conservation program of a critically endangered population of M. adspersa that involves captive breeding, relatedness and paternity analyses, reintroduction, and landscape genetics.