Laboratory assays and field surveys showed that Ascosphaera larvis (Bissett) is a pathogen of alfalfa leafcutting bee larvae, capable of causing high mortality in commercial populations. In one population, over 21% of bees were found to be infected by A. larvis. However, overall levels of the disease are low and it is unlikely that this pathogen poses an immediate threat to commercial leafcutting bee populations in Canada. The LD50 was determined to be 1.9 × 105 spores/bee. Elevated levels of CO2 are required for in vitro spore germination. The disease can easily be diagnosed within bee cells by X-ray radiography, thereby enabling disease levels to be monitored using conventional methods utilized by the industry to monitor leafcutting bee quality.