Objective To acquire the prevalence and molecular identification data on Syphacia muris and provide reference for the revision of national standard. Methods 923 batches of 5199 SPF animals (including one batch of 5 monkeys, 3 batches of 25 mini-pigs, 28 batches of 55 rabbits, 13 batches of 248 hamsters, 37 batches of 198 guinea pigs, 93 batches of 459 rats, 742 batches of 4179 mice, 5 batches of 25 chickens and one batch of 5 ducks) and 145 batches of 1389 clean animals (including one batch of 3 rabbits, 4 batches of 31 hamsters, 16 batches of 157 guinea pigs, 32 batches of 268 rats and 92 batches of 930 mice) came from 50 different manufactures in China. Direct microscopy real-time dynamic video recording techniques in combination with morphological identification method were applied to screen the Syphacia muris infestation. A multiple polymerase chain reaction (multiple-PCR) testing of the isolate based on amplification of the conserved portions of the Syphacia muris internal transcribed spacer (ITS), 28S ribosomal RNA (28S rRNA), NADH dehydrogenase subunits 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and the molecular sequencing of the multiple-PCR amplicons was used to confirm the Syphacia muris infection. Results Syphacia muris eggs, larvae and adults were detected by using direct microscopy real-time dynamic video recording technique. Syphacia muris were detected based on the morphology and size of ovum, larvae, and female and male adult worms. Multiple-PCR and sequencing were performed to identify ITS, 28S rRNA, nad1 and cox1 genes of DNA extracted from the single egg, larva and adult parasite Syphacia muris. This approach allowed the specific identification with no amplicon being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the amplified sequences. Molecular characterization by multiple-PCR amplification and sequencing of the ITS, 28S rRNA, nad1 and cox1 genes demonstrated the presence of Syphacia muris. Multiple-PCR followed by sequencing confirmed 285 of 5199 SPF and 135 of 1389 clean animal samples classified as positive by using direct microscopy real-time dynamic video recording technique in the study as containing Syphacia muris-specific DNA. Comparison of the partial sequences of the ITS, 28S rRNA, nad1 and cox1 genes revealed 100% similarity amongst Syphacia muris from different animals. The prevalence of Syphacia muris infection in SPF and clean animals were 5.5% (285/5199) and 9.7% (135/1389), respectively. Conclusions Direct microscopy real-time dynamic video recording technique, multiple-PCR and sequencing can be used to rapidly detect and accurately identify Syphacia muris. The zoonotic nature of Syphacia muris can be regard as a public health alter, hence the good quality control of animal has an important role in protecting human health and safeguarding people safety. This is the first molecular identification and infection investigation of Syphacia muris in SPF and clean animals in China.