The golden loach (Sabanejewia balcanica) is a species that exhibits significant morphological variability in response to habitat conditions. Since the morphological diversity of the golden loach in Croatia has not been adequately researched, the aim of this research was to determine the morphological characteristics of golden loach populations from 11 sites in the Danube basin rivers and examine whether there are differences among populations. Morphometric analyses proved to be a suitable method for identifying differences among populations, while meristic analyses were not as useful. The results of morphometric analyses indicated that there is a variability among populations and that the populations which are geographically close or have similar ecological habitat conditions tend to cluster together. The most interesting result was the separation of the population from the Sava River, sampled near Slavonski Brod, which significantly differed in most analyzed traits and exhibited a phenotype with a strong resemblance to the morphology of the species S. bulgarica. Morphological characteristics alone are insufficient to determine the species status, and it is necessary to conduct a detailed genetic analysis of the studied populations to determine whether the different morphotypes of the golden loach are influenced by the ecological conditions or potentially represent the presence of a new species for the Croatian ichthyofauna.