Objective: To study the prevalence and genotype of Enterobius (E.) vermicularis from adhesive tape samples in the cities of Shiraz and Khorramabad, Iran. Methods: A total of 1 000 adhesive tape samples from kindergartens in Shiraz (500 samples) and Khorramabad (500 samples) were collected and tested using a microscope to find E. vermicularis egg/s. A questionnaire was filled out for each sample. In order to characterize the genotype of E. vermicularis, the PCR-sequencing method of the mitochondrial cytochrome C oxidase subunit 1 (cox1) gene was used. Genomic DNA was extracted from the positive scotch tape samples of E. vermicularis. The cox1 gene was amplified by the polymerase chain reaction and sequenced. The sequence data were aligned using the BioEdit software and compared with the published sequences in GenBank. Phylogenetic analysis was performed using the maximum likelihood method. Results: The parasitological method showed that 15 out of the 500 samples from Shiraz (3.00%) and 12 out of the 500 samples from Khorramabad (2.40%) were infected with E. vermicularis eggs. BLAST analysis indicated that the sequenced isolates belonged to E. vermicularis genotype B while three different haplotypes were also identified. Conclusions: This is the first study on genotyping E. vermicularis in the cities of Shiraz and Khorramabad. Considering the public health importance of the disease, further studies are necessary to characterize the genotype of E. vermicularis in human populations from other regions of Iran.