Length variation and heteroplasmy were observed in PCR products of the first half of mtDNA control region of both Hong Kong grouper (Epinephelus akaara) and yellow grouper (Epinephelus awoara). DNA sequencing unveiled the phenomena were caused by the presence of species-specific long variable number tandem repeats (VNTRs). This is the first report on the mtDNA VNTRs and their heteroplasmy in groupers. Moreover, these VNTRs are also the longest such structure found in teleost fish. Thereafter, we designed two species-specific PCR reverse primers according to the 3′ end sequences of the VNTRs and successfully established assays for the identification of these two sympatric grouper species.