BACKGROUND: Various flea species have already been reported from dogs, among which the most important ones include Ct. felis, Ct. canis, and P. irritans. Fleas can cause annoyance in dogs and human and transmit a variety of bacterial, fungal, and viral agents to the host. In addition, they could function as an intermediate host of Dipylidium caninum and Hymenolepis diminuta.  OBJECTIVES: Due to the lack of molecular species-associated identification data, we conducted the current study to differentiate Ct. felis and Ct. canis with molecular assay. METHODS: In the present study, 605 fleas were primarily collected from the dogs referred to Tehran Veterinary Faculty hospital. Subsequently, the flea species were identified under a microscope with morphological keys. Afterwards, COX1 genes of Ct. felis and Ct. canis were amplified via PCR and the locus was finally compared utilizing RFLP and sequencing. RESULTS: Totally, 605 fleas were isolated from 20 dogs. In morphological studies, three species were identified: Ctenocephalides felis, Ctenocephalides canis, Pulex irritans. Pulex irritans had the highest frequency (61.8 %). In molecular study, 552 bp fragment of COX1 gene in two species was amplified and seen on agarose gel. After sequencing, it was seen that two species sequences in COX1 locus had a similarity of 99 % and all of them depended on Ct. canis. In PCR-RFLP, in which Taq1 enzyme was used for differentiation of two species, the same result was obtained. CONCLUSIONS: Even though these two species of dog flea are distinct morphologically, their molecular differentiation using COX1 genes was not successful.