Ticks are blood ectoparasites that feed on domestic, wild animals and humans. They spread a variety of infections such as protozoa, viruses, and bacteria. Moreover, cattle reared by smallholder farmers are susceptible to ticks and tick-borne pathogens. Therefore, accurate identification of ticks and detection of tick-borne pathogens is crucial. The main aim of this study was to identify and characterize ticks and tick-borne pathogens from selected villages in Greater Letaba Municipality, Limpopo Province, using morphological and molecular techniques. A total of 233 ticks were collected from cattle and identified morphologically using appropriate morphological keys. The following tick species were identified: Amblyomma hebraeum, Hyalomma rufipes, Hyalomma truncatum, Rhipicephalus appendiculatus, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Rhipicephalus evertsi evertsi, and Rhipicephalus sanguineus. Rhipicephalus spp. was the most common species accounting to 73.8% of the identified ticks. The genomic DNA was extracted from the whole tick for tick identification and from midguts of the ticks for the detection of tick-borne pathogens, followed by amplification and sequencing. A total of 27 samples were positive for tick-borne pathogens: 23 samples tested positive for Theileria and four samples tested positive for Ehrlichia. Anaplasma and Rickettsial OmpB could not be detected from any of the samples. There was no obvious grouping of ticks and tick-borne pathogens on the bases of their locality. The findings of this study confirm previous reports that indicated that cattle reared by smallholder farmers harbor various ticks and tick-borne pathogens of veterinary, public health, and economic importance. Regular monitoring of tick infestations in villages around the study areas is recommended to avoid disease outbreaks.