Ectoparasites not only cause pruritus and dermatitis but also play a crucial role in vector-borne disease in domestic dogs. This study aims to investigate the prevalence of ectoparasite infestation and the molecular characteristics of ticks and fleas. A cross-sectional study was conducted on 720 dogs in two provinces and one city in the Mekong Delta of Vietnam: An Giang, Can Tho, and Kien Giang. The identification of ectoparasites was performed based on parasitological methods, conventional PCR, and sequencing. The findings revealed that domestic dogs were infected ectoparasites with a high infection rate of 57.92% (417/720). Age, lifestyle of dogs, and veterinary management practices of dog owners had a significant influence on the ectoparasite infestation in the surveyed dogs. Six different species of ectoparasites were identified by morphological characteristics, including ticks (Rhipicephalus sanguineus), fleas (Ctenocephalides canis, Ctenocephalides felis felis), demodectic mange (Demodex canis), louse (Trichodectes canis), and mites (Sarcoptes canis). In this study, ticks (R. sanguineus) were the most common ectoparasites in infected dogs (59.95%), followed by fleas (Ctenocephalides spp.) at 34.77%, Trichodectes canis at 6.71%, Demodex canis at 2.63%, and Sarcoptes canis 1.92%. Analyzing the 16S rDNA sequence of R. sanguineus in domestic dogs in the Mekong Delta showed high homologs to the R. sanguineus strains from Brazil and India. Furthermore, the ITS-1 sequence of Ctenocephalides revealed a close relationship to the C. canis isolates in Spain. Keywords: Domestic dogs, ectoparasites, Mekong Delta, molecular characteristics