Environmental DNA (eDNA) metabarcoding has emerged as a potentially powerful tool to monitor invasive fish species.As an alternative (or complementary) tool for biodiversity monitoring,eDNA metabarcoding had been used to detect species in aquariums,which represents an important transit avenue for introducing non-indigenous species with high population densities.In this study,eDNA metabarcoding as well as morphological characterization were used to reveal the diversity of non-indigenous species in a large aquarium at Qingdao Underwater World.Environmental DNA metabarcoding of 14 water samples at five locations from the Big Water Tank detected 24 non-indigenous species and four putative non-indigenous operational taxonomic units (OTUs).In contrast,only 20 non-indigenous species were observed by morphological characterization.Some species undetected by morphological characterization,such as Oreochromis niloticus (Linnaeus,1758),are highly adaptable to various environments and/or have invaded preferred regions where they threaten native aquatic species.eDNA metabarcoding also detected seven local fishes that were not identified by morphological characterization.However,analysis of OTU diversity among stations and sample replications revealed that eDNA varied within and/or between stations.Increasing sampling effort as well as negative controls are required to increase the detection rate of species and to eliminate false-positive OTUs.