A molecular method is applied for differentiating the morphologically related leafminers Liriomyza trifolli and L. sativae on tomato cultivation. The method requires multiplex polymerase chain reaction (PCR) amplification of a region of mitochondrial cytochrome oxidase DNA using multiprimer sets. The method divides the mitochondrial fragment of L. trifolli into two fragments and L. sativae into three fragments. It is faster, less costly, and easier than random amplified polymorphic DNA-PCR, PCR-restriction fragment-length polymorphism, and DNA sequencing and more sensitive than the enzyme electrophoresis method, which are other ways to differentiate these two species. We applied the method to samples from populations of another place, sex, and stage and obtained the same results.