The genus Cnephasia is represented by more than 70 species of insects worldwide, including serious pests of agricultural plants, mainly cereals. Since members of this genus are frequently very similar externally, species determination based on morphotaxonomy is time-consuming and difficult, especially for non-taxonomists. Hence, it could possibly be replaced by molecular biology approaches. A short nucleotide sequence of mitochondrial cytochrome oxidase I (mtCOI) constitutes a commonly used molecular marker for phylogenetic analyses identification. The aim of this work was molecular species determination of leaf rollers, collected in Poland, that on the basis of external features were hardly distinguishable. We amplified, sequenced and phylogenetically studied the fragment of the mtCOI gene for each individual. Comparative analyses showed the highest nucleotide similarity to C. genitalana, C. longana, C. pasiuana, C. asseclana and C. stephensiana, which was also confirmed by phylogeny. Obtained results showed genetic variation of the analyzed fragment of the mtCOI gene between analyzed Cnephasia spp. found in Poland that can be helpful in proper species determination. This in turn, may be essential for successful biological control and pest monitoring in crop cultivation.