In the present study we examined and compared external morphology as well as osteological characters of two species of the genus Ameiurus (Teleostei, Ictaluridae): the black bullhead (Ameiurus melas) and brown bullhead (Ameiurus nebulosus), two catfish species that are invasive to Europe. In total 83 (53 and 30) individuals were captured in the Czech Republic and the Slovak Republic and further analysed. For genetic screening of both Ameiurus species were chosen three DNA regions: part of nuclear Recombination activating gene 1 (RAG-1), mitochondrial gene for cytochrome b (cyt b) and mitochondrial sequence which includes 12S and 16S ribosomal RNA genes. The results presented in this paper concern specific osteological characteristics (original scheme of pectoral spine and pectoral girdle) used for the determination purposes and also verification that mathematical multivariate methods for external morphology analysis can be used for the determination of Ameiurus species. The summary of 80 out of 83 fish (96.4 %) was correctly classified using the discriminant analysis.