Mislabeling fish products is a crucial and growing issue concerning public health and economic concerns in the global fish business. Although DNA barcoding is an effective sequencing-based approach for identifying fish species adulteration, it is sometimes difficult to amplify PCR products with full-length barcode biomarkers (~650 bp) because of DNA degradation, particularly in extensively processed fish items. On the other hand, short-length barcodes showed higher stability under severe food processing treatments. Consequently, mini barcodes would be a suitable substitute for full-length DNA barcodes for identifying species in DNA degraded processed fish products. Therefore, this study aimed to explore the usefulness of DNA barcoding for identifying different types of fish products by developing a medium-sized mini barcode. We developed a set of universal primers targeting the mitochondrial 16s rRNA gene to amplify a 198-bp sequence to detect fish species in processed fish products. The designed universal primers set showed high specificity, amplifying only tested fish species (38 species), and no PCR products were found from 22 other non-target species (animals and plants). During the specificity evaluation, eukaryotic endogenous control was utilized to exclude the possibility of any false-negative detection. In addition, the validation of the developed primers set was performed on various heat-treated fish samples (boiled, autoclaved, and microwaved) and shown to be highly stable under all processing treatments. As a result, 92% of the analyzed commercial fish products were successfully identified with 96–100% sequence similarities. The developed universal biomarker is a useful tool for detecting fish species in highly processed foods, and it could be beneficial in identifying fish species replacement, protecting consumers’ health and financial interests.