Piroplasmosis is a disease of domestic and wild animals caused by tick-borne protozoa of the genera Theileria and Babesia. Piroplasmosis leads to substantial economic losses in the livestock industry. This disease has been frequently reported in subtropical and tropical regions worldwide. However, information regarding the prevalence of piroplasma in ticks collected from dogs and cattle is lacking in most areas. To assess the potential threat of piroplasmosis in South China, 671 ticks were collected in Guangxi Province. Deoxyribonucleic acid (DNA) was extracted from these ticks to evaluate the presence of piroplasma through a reverse line blot (RLB) hybridization assay using the hypervariable V4 region of the piroplasmic 18S ribosomal ribonucleic acid (r)RNA genes as probes to detect Theileria and Babesia species. The RLB results indicated that 144/671 (21.46%) ticks were infected with piroplasma species belonging to the genera Theileria or Babesia. Theileria buffeli, Babesia vogeli, Theileria annulata, and Theileria luwenshuni were present in the ticks at frequencies of 60/671 (8.94%), 21/671 (3.13%), 6/671 (0.89%), and 6/671 (0.89%), respectively. Mixed infections with 2 or more piroplasma species were present in 8/671 (1.19%) tick samples. The hypervariable V4 region of the piroplasmic 18S rRNA genes from 6 tick DNA samples with single infections was cloned, sequenced, and aligned to related sequences from GenBank. The Theileria and Babesia sequences were analyzed separately. 18S rRNA gene fragment sequences of T. annulata and T. buffeli were compared with previously reported homologous sequences. All 3 B. vogeli sequences examined in this study were grouped into the same cluster and belonged to the same genotype. The present study provides important epidemiological information regarding piroplasmosis occurrence in China. The existence of tick-borne piroplasma likely leads to high infection risks among the local animals in the studied areas.