In mammals, the toll-like receptors (TLRs) play a major role in initiating innate immune responses against pathogens. Comparison of the TLRs in different mammals may help in understanding the TLR-mediated responses and developing of animal models and efficient therapeutic measures for infectious diseases. The Chinese tree shrew (Tupaia belangeri chinensis), a small mammal with a close relationship to primates, is a viable experimental animal for studying viral and bacterial infections. In this study, we characterized the TLRs genes (tTLRs) in the Chinese tree shrew and identified 13 putative TLRs, which are orthologs of mammalian TLR1-TLR9 and TLR11-TLR13, and TLR10 was a pseudogene in tree shrew. Positive selection analyses using the Maximum likelihood (ML) method showed that tTLR8 and tTLR9 were under positive selection, which might be associated with the adaptation to the pathogen challenge. The mRNA expression levels of tTLRs presented an overall low and tissue-specific pattern, and were significantly upregulated upon Hepatitis C virus (HCV) infection. tTLR4 and tTLR9 underwent alternative splicing, which leads to different transcripts. Phylogenetic analysis and TLR structure prediction indicated that tTLRs were evolutionarily conserved, which might reflect an ancient mechanism and structure in the innate immune response system. Taken together, TLRs had both conserved and unique features in the Chinese tree shrew.