Speciation genes are responsible for genetic incompatibilities in hybrids of incipient species and therefore participate in reproductive isolation leading to complete speciation. Hybrid males between Drosophila melanogaster females and D. simulans males die at late larval or prepupal stages due to a failure in chromosome condensation during mitosis. However a mutant male of D. simulans, named Lethal hybrid rescue (Lhr), produces viable hybrid males when crossed to females of D. melanogaster. Recently the Lhr gene has been proposed as corresponding to the CG18468 gene in D. melanogaster. However this identification relied on sequence characteristics more than on a precise mapping and the use of the GAL4/UAS system to drive the transgene in D. melanogaster might have increased the complexity of interaction. Thus here we propose an independent identification of the Lhr gene based on a more precise mapping and transgenic experiments in D. simulans. We have mapped the Lhr gene by using Single Nucleotide Polymorphisms (SNPs) and identified within the candidate region the gene homologous to CG18468 as the Lhr gene as it was previously reported. Transgenic experiments in D. simulans with the native promoter of CG18468 prove that it is the Lhr gene of D. simulans by inducing the lethality of the hybrid males.