Round goby Neogobius melanostomus is one of the three invasive Ponto-Caspian gobies found in the Sava River basin in Croatia. It has a negative effect on the native ecosystems and is more abundant in highly polluted areas. The objective of this study was to determine the fluctuating asymmetry of four morphometric characters, as well as otolith width and length. Thirty individuals of N. melanostomus were collected and analysed at two sampling sites in the main course of the Sava (Babina Greda, Slavonski Brod) and in a modified habitat (Sava-Odra canal). The squared coefficient of asymmetry variation was used to analyse potential differences between populations. We identified variations in the asymmetry values of the analysed characters, as well as otolith width and length between sampling sites. The highest fluctuating asymmetry was observed in individuals at the Babina Greda sampling site, which could be due to high environmental stress. The results of the study show that the study of fluctuating asymmetry can be used on invasive fish species as a simple approach to identifying habitat quality.