Abstract: In order to establish a simple and rapid method for the differential detection of Canine distemper virus（CDV） wild strains and vaccine strains, in this experiment the differential detection primers were designed based on the H gene sequences of CDV wild strains and vaccine strains in GenBank, and one-step RT-PCR was established for CDV wild strains and vaccine strains, which verified the specificity, sensitivity, repeatability and compliance of the method. The established double one-step RT-PCR method of CDV wild strains and vaccine strains was used 505 throat swab samples and disease material samples of infected dogs collected from 11 different cities in Jiangsu Province from 2017 to 2020 were detected, to determine the clinical applicability of double one-step RT-PCR of CDV wild strains and vaccine strains. The results showed that the method could amplify the specific bands of 477 bp, 677 bp, 477 bp/677 bp for CDV wild strains, CDV vaccine strains, CDV wild/vaccine strain, respectively, while the detection results of Canine parvovirus（CPV）, Canine adenovirus（CAV）, Canine parainfluenza virus（CPIV）, and Canine coronavirus（CCV） were all negative. The detection sensitivity of double one-step RT-PCR was 23.1 pg RNA; the coincidence rate of double one-step RT-PCR and RFLP method was 100%; the results of double one-step RT-PCR intra-assay and inter-assay repeatability were completely consistent. Using this method to detect 505 clinical samples, the positive rates of CDV wild strains and vaccine strains were 8.91% and 72.48%, respectively. The results suggested that the established one-step RT-PCR of CDV wild strains and vaccine strains had good specificity, sensitivity, repeatability, accuracy and clinical applicability.