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To characterize ticks in cattle from Guangdong Province and Guangxi Zhuang Nationality Autonomous Region, Southern China, 783 cattle in four localities were examined. Among them, 232 (29.63%) cattle were positive for tick infection. A total of 503 ticks collected in these cattle were further investigated. Two Rhipicephalus species, namely R. microplus and R. sanguineus, were firstly identified by morphological features. Thereinto, R. microplus is the prevalent species in cattle in southern China, with high prevalent in summer and autumn annually. Mixed infection of R. microplus and R. sanguineus was just found in yellow cattle. To further confirm the morphological identification of these cattle-origin ticks, a phylogeographic analysis inferred from the sequences of the ribosomal internal transcribed spacer-2 (ITS-2) was performed, and R. microplus and R. sanguineus were identified. However, the morphological taxonomy of R. microplus has been challenged in recent years. The mitochondrial cytochrome c oxidase subunit 1 (cox1) marker was then used to provide higher resolution of R. mircoplus complex. The re-constructed cox1 phylogenetic tree further identified these R. mircoplus tick samples as R. microplus Clade A. These findings illustrated the prevalence and characterization of cattle-origin ticks in Southern China for the first time, and provided base-line information for further control of tick and tick-borne disease in these areas.