Negative impacts of introduced non-native freshwater species on native species have been increasingly recognized in the world as well as in Turkey. However, there has been relatively little attention on genetic characterization of alien freshwater fishes in their non-native distribution range and virtually no study has been conducted in Turkey despite its crucial importance in invasion biology. The purpose of this study was to elucidate genetic diversity of common non-native freshwater fish species (Carassius auratus, Carassius gibelio, Gambusia holbrooki, Lepomis gibbosus, and Pseudorasbora parva) using mitochondrial Cytochrome c oxidase subunit I (COI) sequences; known as DNA barcodes. Through the whole dataset, seventeen haplotypes (haplotype diversity = 0.8908) were found containing 145 COI sequences. Mean Kimura two-parameter genetic distances were calculated as 0.209 for interspecific distance and 0.009 for intraspecific variation. COI barcode diversity among populations of the same species was found to be low, especially for C. gibelio, G. holbrooki, and L. gibbosus populations which were 0.5%, 0.6%, and 0.3%, respectively. Our results clearly demonstrate the effectiveness of the DNA barcoding approach both for identifications at species level and revealing intraspecific variation among populations, which could be used for effective management measures for invasive species and conservation strategies for indigenous and endemic species.