A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to check the differences of D-loop segment of mitochondrial DNA in the all-male hybrid tilapia progeny, sampled from the aquatic genetic resource bank at the Freshwater Aquaculture Research Center (FARC) and a private fish farm, respectively, in order to elucidate the relationship between the mitochondrial DNA’s maternal heredity and the male ratio of hybrid tilapia progeny.After the digestion of mitochondrial D-loop PCR fragment by different restriction enzymes, the electrophoretic analysis showed that the maternal inheritance of D-loop in the hybrid progeny, sampled from the aquatic genetic resource bank at the FARC, were all identified as ‘Nile tilapia (Oreochromis niloticus) type’, while those sampled from the private fish farm were identified as three categories of ‘Nile tilapia type’, ‘Blue tilapia (O. aureus) type’ and ‘Mozambique tilapia (O. mossambicus) type’.From the above mentioned results and appearance of the hybrid offspring, it was concluded that mother of ‘Blue tilapia type’ hybrid progeny was Blue tilapia, not Nile tilapia. Similarly, mother of ‘Mozambique tilapia type’ was not Nile tilapia. The impurity of maternal broodstock might be the possible reason of the decreasing male ratio of hybrid tilapia progeny in the private fish farm in recent years. In this aspect, it’s important for tilapia aquaculture to increase stable and high ratio of all male hybrid tilapia progeny by management and maintenance of the purity of broodstock.