A high priority in rose (Rosa spp.) breeding research is the transfer of disease resistance, especially to black spot (Diplocarpon rosae Lib.), from wild diploid Rosa species to modern rose cultivars. To this end, amphidiploids (2n = 4x = 28) were induced with colchicine from five interspecific diploid (2n = 2x = 14) hybrids involving the black spot resistant diploid species R. wichuraiana Crép, R. roxburghii Thratt., R. banksiae Ait., R. rugosa rubra Hort., and R. setigera Michaux. Two application procedures (agitation of excised nodes in colchicine solution or tissue culture of shoots on medium with colchicine), five colchicine concentrations (0.0, 1.25, 2.50, 3.76, and 5.01 mmol), and five durations (2, 3, 5, 8, and 10 d) were used. After colchicine treatment, the materials were cultured in vitro and the surviving explants were examined for the “gigas” characteristics typical of doubled diploids. Chromosome counts of morphologically suspect genotypes confirmed 15 amphidiploids among 1109 plants that survived colchicine treatment. Although the effect of colchicine treatment varied some among interspecific hybrids, 2.50 mmol for 48 h of node agitation or 1.25 mmol for at least 5 d of shoot culture were optimal.