Fish substitution in fish products is an important issue in fish markets, as it is a widespread practice. An authentication protocol for Rohu, Thaila and Tilapia was developed by multiplex PCR. Three species-specific and one degenerate common forward primer were designed using the Cytb gene of the mitochondrial genome. These primers for Labeo rohita, Labeo catla and Oreochromis niloticus showed the fragment size of 235 bp, 186 bp and 506 bp on the agarose gel, respectively. The primers for L. rohita and L. catla were sensitive to 0.1 ng of DNA template, while for O. niloticus this value was 1 ng of DNA template. A total of 230 commercial samples (160 fried and 70 processed fish products) were screened, where 60% mislabeling in fried and 30% mislabeling in processed fish were found. This multiplex PCR protocol could give useful insights for food inspection and enforcement of regulatory food control.