Objective To identify the prevalence of ECTV in mice. Methods A sensitive and specific FQ-PCR assay for ECTV was developed and used to detect and quantitate ECTV in mouse samples by analyzing the sequence of the crmD gene in ECTV. Results This assay detected a minimum of 10 copies of standard DNA. The assay was applied to 63 naked mole rat spleen samples and 22 mouse spleen samples raised in the laboratory, which provided negative result . In tissue samples from four ground squirrels raised in our laboratory, the positive rate was 50%. Some positive samples amplified with primers for the crmD gene had 99% similarities to ECTV as determined by sequencing the PCR products. Conclusions Our result suggest that routine surveillance of ECTV in laboratory mice cannot be ignored.