As an important adaptor protein in the intracellular signaling of Toll-like receptor superfamily, myeloid differentiation factor 88 (MyD88) plays a crucial role in the innate immune response. The full cDNA sequence of MyD88 (GenBank Acession number: JN032017) has been isolated by RACE-PCR method in Tilapia(Oreochromis aureus). The MyD88 gene is 1 611 bp, including a 155 bp 5'-terminal untranslated region (UTR), a 589 bp 3'-UTR and an 867 bp open reading frame (ORF), which encodes a polypeptide of 288 amino acids. The MyD88 protein contains the typical TLR and IL-1R-related (TIR) domain and death domain (DD). Homology analysis revealed that the predicted amino acid sequence of Oreochromis aureus MyD88 shared the highest identity 85.8% to Siniperca chuats, 70%-82% to other fishes, and 63%-66% to mammals. The phylogenetic tree based on MyD88 proteins from 15 species showed that O.aureus shared the closest relationship with Siniperca chuatsi and Larimichthys crocea, all pertaining to Perciformes. Expression of MyD88 was determined by real-time RT-PCR (qRT-PCR) in different tissues. The results revealed that MyD88 transcripts were expressed in all tested tissues, being strongly expressed in the ovary, moderately expressed in intestine, spleen, liver, kidney, gill and blood, and weakly expressed in muscle and testis. This study will lay a foundation for further exploring the role of MyD88 in the TLR signaling in Tilapia.