The present study was conducted to determine the effect of the D genome on embryoid induction and green plant regeneration in wheat anther culture and how it is influenced by low temperature and mannitol treatment. For this reason, the anther culture response of two Canadian bread wheat cultivars and their extracted tetraploids (AABB) was studied. As controls two cultivars well responding to anther-culture (i.e. cvs. Kavkaz/Cgn and Acheron) and a no-responding cultivar (cv. Vergina) were used. Approximately 3000 anthers of these cultivars were cultured and three pre-treatments were applied: cold pre-treatment for 7 and 18 days at 4°C, and 0.3M mannitol for seven days at 4°C. W14 and 190-2 were used as induction and regeneration media, respectively, and the basic MS medium as the rooting medium. No green plants were produced from the tetraploids, which supports the view that the D-genome chromosomes are necessary for androgenic response in wheat. Furthermore, the Canadian cultivars performed better after 18-day pre-treatment at 4°C. The extracted tetraploids produced fewer embryoids and performed better after seven days of cold pre-treatment. The controls well responding to anther culture performed better than the Canadian cultivars, although their best response was recorded after seven-day cold pre-treatment. Cultivar Vergina produced no green plants. The presence of mannitol influenced negatively both embryoid and green plant production. It was concluded that the D genome plays a crucial role in anther culture response of wheat and that this response is influenced by both the genotype and the duration of cold pre-treatment.