The increase in seafood demand resulted in frequent seafood frauds such as species mislabeling, which could lead to health risks or financial deprivation of consumers. Applicable assays for species identification would result in seafood fraud prevalence reduction in the fishing industry. In this study, we examined the application of high sensitivity DNA-based assays for the detection of species substitution for 82 specimens isolated from 15 seafood samples containing squid (order: Cephalopoda). Fragments of COI and 28S were used to assess whether commercially important species could be identified using DNA based approach. Pairwise intra- and inter-specific p-distances were assessed to determine the possibility of distinguishing L. Vulgaris from other commercially important squid species. We used COI sequences from publicly available databases and sequences determined from reference samples (A. media and L. Vulgaris species) in this study. Employees of the National Institute of Biology (NIB) performed loop-mediated isothermal amplification (LAMP) specific for L. Vulgaris, which showed no application at the current stage of the study. Phylogenetic analysis was performed as part of DNA barcoding by forming a maximum likelihood tree and a Kimura 2-parameter model in the MEGA–X program. We conclude greater reliability and applicability of the COI region compared to the 28S region, given the higher success in sequencing, the presence of a larger number of reference sequence data in publicly available databases, and a clearer distinction of species using the bootstrap method. Of the 69 successfully identified seafood species analyzed in this study, there are 8.70% of specimens in which species substitution fraud is present. A meta-analysis with a larger number of specimens from the entire Slovenian market is necessary, given that this research analyzed a relatively small number of samples based on which it is difficult to assess the presence of fraud in the entire Slovenian market.