The Fish Invasiveness Scoring Kit (FISK) is currently one of the most popular pre-screening tools for freshwater fishes. A recent upgrade has ensured its wider climatic relevance to countries with subtropical regions. This enhancement is of particular importance to Australia, which encompasses tropical, arid, and temperate zones, and where the introduction of non-native fish species poses a significant risk to biodiversity. In this study, 55 fish species previously evaluated in a U.K.-based calibration of FISK are reassessed for their potential invasiveness in the Murray-Darling Basin (MDB; southeastern Australia), the continent's largest catchment encompassing arid and temperate climates. Approximately half of the species were classed as “medium risk” and the other half as “high risk,” and the ≥19 threshold previously identified from the calibration study was confirmed. The three highest scoring species (common carp Cyprinus carpio carpio, goldfish Carassius auratus, and eastern mosquitofish Gambusia holbrooki) were those already present and invasive in the area, whereas nearly half of the tropical and subtropical species had lower scores compared to U.K. assessments, possibly because of climate change predictions of drier conditions across the MDB. There were some discordances between FISK and two Australian-based assessment protocols, one of which is qualitative and the other represents a simplified version of FISK. Notably, the Australian origins of FISK should provide for an additional reason for further applications of the tool in other RA areas (i.e., drainage basins) of the continent, ultimately encouraging adoption as the country's reference screening tool for management and conservation purposes.