Double-haploidization (DH) is one of the favorable techniques to obtain 100% pure double haploid plants (DH’s) for generating the new F1 cultivars in a short time. The fecundity of this technique depends on the high quantity of haploids and also fertile DH’s. However, there are no comprehensive reports on the chromosome doubling and fertility (fruit and seed-set) of winter squash and pumpkin haploids, currently. Thus, to obtain high frequency and fertile DH’s, the efficiency of different anti-mitotic agents (colchicine, ami-prophos methyl, trifluralin and oryzalin) was tested at various concentrations and exposure times for both in vitro and in vivo conditions. Haploid plantlets recovered from anther cultures of winter squash and pumpkin lines were used for DH program. The haploid plants were wholly immersed in aqueous solutions of anti-mitotic agents in vitro and apical parts of haploid plantlets were treated with anti-mitotic agents three times in vivo. Since some plants remained haploid, and the highest DH efficiency was obtained from multiple treatments of colchicine to shoot tips of haploid plants in the rate of 93.3%. In vivo multiple treatments of 1% colchicine for an hour was found to be the best doubling procedure for the recovery of high-frequency fertile DH’s in our winter squash and pumpkin breeding program.