Abstract: The target region amplification polymorphism (TRAP) marker is a novel functional molecular marker. In present study, we compared the optimization of the reaction system for TRAP markers in tilapia (Oreochromis spp.) between L16(45) orthogonal design and single-factor design. The results of the two approaches showed that the concentrations of dNTPs, random primer and DNA were the same, while those of Mg2+ and TaqDNA polymerase dosage varied, and interaction among different factors were observed. The orthogonal design is simpler, more scientific and reasonable than single factor design. The optimal TRAP reaction system for tilapia includes 60 ng DNA template, 1.5 mmolL-1 Mg2+, 0.3 mmolL-1 dNTPs, 0.5 U TaqDNA polymerase, 7 pmolL-1 random primers and 10 pmolL-1 fixed primer. The stability and repeatability of the reaction system had been verified in 4 tilapia populations. The TRAP marker provides new insights into evaluation of genetic diversity, germplasm identification, molecular marker-assisted breeding and other related researches of tilapia.