Technical Abstract: Chalkbrood is a disease of bee larvae caused by fungi in the genus Ascosphaera. The alfalfa leafcutting bee (Megachile rotundata) is managed for alfalfa seed production. Chalkbrood is a serious mortality factor in the managed bees, especially in the U. S. A. aggregata is thought to be the main species causing these infections, but at least nine other species occur in megachilids, and we have frequently seen them in M. rotundata populations. We have developed a molecular marker that allows us to identify the presence of any of these species when they occur in bee larvae, regardless of whether symptoms are yet apparent. Spores are usually required for morphological identification, but some of the Ascosphaera (including A. aggregata) do not complete development on artificial media. Therefore, when using morphological methods for identification, sporulating cadavers are necessary for species identification. We have developed a restriction analysis method, using cleaved amplified polymorphic sequences, to identify to species those Ascosphaera that are associated with M. rotundata.