Methods for the rapid detection of the presence or absence of Babesia in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers and probes targeting Babesia and kits are provided that are designed for the detection of Babesia, including, but not limited to, the Babesia species of B. microti, B. divergens, B. duncani, and B. venatorum. Also described are kits, reaction mixtures, and oligonucleotides (e.g., primer and probe) for the amplification and detection of Babesia.