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Babesia bovis is a causal agent of bovine babesiosis, a disease which leads to mortality and morbidity and impacts the cattle industry worldwide. We amplified, cloned and sequenced the B. bovis merozoite surface antigen-2b (msa-2b) gene (_∼940 bp) and the near full-length 18S rRNA gene (_∼1600 bp) from cattle samples from South Africa and Mozambique to determine sequence variation between B. bovis parasites in the region. A TaqMan quantitative real-time PCR (qPCR) assay (18S rRNA gene) was optimised for the detection of B. bovis and estimation of parasitaemia in field samples from cattle from southern Africa. Phylogenetic analysis grouped the Msa-2b sequences in six clades and these were 59.7 to 99.6% identical to reference sequences. Sequence variation amongst B. bovis 18S rRNA sequences was found at 2 to 36 positions, and the sequences were 97 to 99% identical to published sequences …