The genomic locus and cDNA encoding Babesia bovis dihydrofolate reductase-thymidylate synthase (DHFR-TS) were cloned and sequenced. A single dhfr-ts gene, composed of four exons, encodes a 511 aa protein that is most closely related to Plasmodium falciparum DHFR-TS. The genomic locus is characterized by the presence of four other genes of which at least three are expressed during the erythrocytic cycle. Three of the genes were highly conserved in closely related Theileria species and for two of the genes and dhfr-ts, gene synteny was observed between B. bovis and Theileria parva, B. bovis in vitro cultures displaying approximately 10-20-fold decreased sensitivity towards the antimalarial drugs WR99210 and pyrimethamine were selected repeatedly after prolonged growth in presence of drugs. Five cultures examined in detail were shown to encode a DHFR-TS carrying amino acid substitution S125F. Three-dimensional-modelling, using the P. falciparum DHFR structure as a template, suggests that substitution S125F protrudes into the binding site of NADPH. The S125F mutant could be isolated by growth under pyrimethamine or WR99210 pressure conferring cross-resistance to both drugs. Although opposing selection for pyrimethamine or WR99210 resistance was reported recently using P. falciparum or P. vivax strains carrying wildtype dhfr, the results obtained here are reminiscent of a quadruple mutant of P. falciparum dhfr displaying strong resistance to pyrimethamine and 10-fold enhanced resistance against WR99210. Wildtype B. bovis DHFR carries three mutations present in this mutant possibly explaining the low sensitivity to pyrimethamine and the ease by which moderately WR99210 resistant mutants could be isolated.