Rhipicephalus microplus and R. decoloratus are one-host ticks that preferentially feed on cattle. They are capable of transmitting various tick-borne pathogens which may be detrimental to the agricultural and livestock industry in South Africa. Previous studies have shown that R. microplus forms five lineages in the R. microplus complex, segregating into different geographical areas based on mitochondrial markers. This study examined the phylogenetic relationship within and between R. microplus and R. decoloratus using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes. The results showed that the nuclear ITS2 marker is informative for interspecific variation but lacks the resolution for intraspecific variation. Analysis of the mitochondrial COI gene revealed that R. microplus ticks from South Africa grouped into a clade comprised of ticks from Asia and South America. The population structure of these two tick species was also investigated using novel microsatellite markers. Population structure analyses revealed that both the R. microplus and R. decoloratus populations presented with two genetic clusters. Rhipicephalus microplus ticks from the Kwa-Zulu Natal (KZN) province belonged to cluster 1, and those from the Eastern Cape (EC) province predominantly grouped into cluster 2. No observable population structure was noted for R. decoloratus. The overlap of genetic clusters in both species could be attributed to inbreeding between the regions by unrestricted movement of cattle across provinces. Such movement promotes tick mobility, gene flow and the homogenisation of tick populations.