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Description: Adequate species’ identification is critical for the detection and monitoring of biological invasions. In this study, we proposed and assessed the efficiency of newly created primer sets for the genetic identification of non-indigenous species (NIS) of fishes in the Volga basin based on: (a) a “long” fragment of cytochrome c oxidase subunit one of the mitochondrial gene (COI) (0.7 kb), used in “classical” DNA barcoding; (b) a short 3’-fragment (0.3 kb) of COI, suitable for use in high-throughput sequencing systems (i.e., for dietary analysis); (c) fragment of 16S mitochondrial rRNA, including those designed to fill the library of reference sequences for work on the metabarcoding of communities and eDNA studies; (d) a fragment of 18S nuclear rRNA, including two hypervariable regions V1-V2, valuable for animal phylogeny. All four sets of primers demonstrated a high amplification efficiency and high specificity for freshwater fish. Also, we proposed the protocols for the cost-effective isolation of total DNA and purification of the PCR product without the use of commercial kits. We propose an algorithm to carry out extremely cheap studies on the assessment of biological diversity without expensive equipment. We also present original data on the genetic polymorphism of all mass NIS fish species in the Volga-Kama region. The high efficiency of DNA identification based on our primers is shown relative to the traditional monitoring of biological invasions.