Virological testing and monitoring is a fundamental part of quality control of experimental animals. However, there are few papers regarding the spectrum and status of natural infection in wild tree shrews with human and animal pathogenic viruses. Using enzyme-linked immunosorbent adsorption assay (ELISA), we tested sixty wild tree shrews captured from Qinglong, an outskirt region of Kunming, Yunnan Province, China for eleven viruses, including herpes simplex virus, coxsackie virus, influenza virus, HAV, HBV, HCV, HDV, dengue virus, hemorrhagic fever virus and measles virus. Our results showed that, in the serum samples, 22/60 (36.7%) and 1/60 (1.67%) were antibody positive for herpes simplex virus and coxsackie virus, respectively, and 4/60 (6.7%) were antigen positive for rotavirus in the feces. The remaining species of viruses were negative in these tree shrews. Based on these results, we propose that herpes simplex virus, coxsackie virus and cotavirus should be listed as top priority for routine virological monitoring of tree shrews.