Blossom blight of alfalfa, caused by Botrytis cinerea and Sclerotinia sclerotiorum, can be an important constraint to alfalfa seed production on the Canadian Prairies. Botrytis cinerea is the predominant pathogen in rain-fed production areas across the northern Prairie region. This study assessed the optimum conditions for infection of alfalfa florets by B. cinerea, and susceptibility in alfalfa based on cultivar and flower colour/orientation. The disease reaction of the cultivars was assessed in detached and intact inflorescences inoculated under controlled conditions and in field trials. The optimum temperature for infection was 20°C, with a minimum of 12 h of surface wetness. Increased duration of surface wetness generally increased infection, except at 30°C, where continued exposure to high temperature reduced infection. There were small but consistent differences in flower infection among cultivars, which were consistent across the testing protocols (detached inflorescence, whole-plant and field). Upward-facing inflorescences had a lower incidence of infection than downward-facing inflorescences. Also, purple florets were slightly less susceptible relative to white/yellow florets, but in only one of the three cultivars assessed. Knowledge of infection requirements from this study can be used to improve management of blossom blight, but the differences among cultivars were generally too small to have an important impact on disease management in the field.