Technical Abstract: The sudden death of several cattle at the beginning of an experiment involving an infestation with Boophilus microplus led to an investigation into the reasons for the mortality. Evidence for Babesia bigemina infection was found in blood smears from the affected animals and a PCR assay was designed to detect the presence of B. bigemina and Babesia bovis in all B. microplus strains maintained at the laboratory. The assay utilizes a nested PCR approach with the first PCR amplifying a segment from the 18S ribosomal RNA gene, including the segment from both B. bigemina and B. bovis but not B. microplus, and a second nested PCR with species-specific primers and annealing temperatures enabling amplification of the 18S ribosomal RNA gene fragment specific to either B. bigemina or B. bovis. We detected the presence of B. bigemina in all strains of B. microplus currently reared at the laboratory and, using archived frozen larval samples from previous generations, documented that the infections had been present for many generations. Using groups of 50 larvae and a rapid DNA preparation protocol involving grinding the frozen tick larvae in PCR buffer and boiling the mixture for 5 minutes, the assay sensitivity allowed the detection of the equivalent of a single infected tick. B. microplus eggs were also analyzed, but results were inconsistent with the crush and boil DNA protocol and the use of a proteinase K digestion-based DNA purification method was necessary to produce consistent results with eggs.