Rooting of single-node leafy stem cuttings of once-blooming rose cultivars (‘Duchesse d’Angoulême’, ‘Hurdals’, ‘Maiden’s Blush’, ‘Mousseuse Rouge’, ‘Polstjårnan’, and ‘Semiplena’) was performed, without the use of rooting stimulants, in two rooting substrates: peat-perlite and peat-sand (1 : 1). The shoots of stock plants were cut at four phenological stages: flower buds closed, flowers open, and immediately after and 7-14 days after petal shedding. The effect of the phenological stage of shoots and rooting substrate was studied on rooting of cuttings, cuttings with retained stock plant leaf, cuttings that create a new shoot, fresh weight of the root system and aboveground part (leaves and shoots), the length of roots and new shoots, and the total leaf area. The proper phenological stage of shoots holds the key for the success of the rooting and growth parameters of cuttings. The four researched cultivars with closed flower buds had significantly higher rooting (in peat-sand: ‘Maiden’s Blush’ – 60.0%, ‘Mousseuse Rouge’ – 75.0%, ‘Polstjårnan’ – 99.4%, and ‘Semiplena’ – 90.0%; peat-perlite: ‘Hurdals’ – 67.5% and ‘Maiden’s Blush’ – 50.0%) and showed good results in percentage of cuttings with new shoots and total leaf area. The possibility of using cuttings acquired from shoots in other stages depended on the cultivar. ‘Maiden’s Blush’ (peat-perlite – 12.9%, peat-sand – 0.7%) and ‘Semiplena’ (peat-perlite – 30.0%, peat-sand – 52.5%) derived from shoots 7-14 days after petal shedding showed a low ability to root, whereby they were diminished in quality. In ‘Hurdals’, these cuttings did not root. The selection of the phenological stage of shoots that will generate a high percentage of rooting does not always correspond to the quality of rooted cuttings. The use of peat-sand as a rooting substrate had a beneficial effect on the efficiency of cuttings. The results of this experiment can be used in ecofriendly cultivation and propagation, according to the European Union Council Directives (no. 91/414/EEC, 79/117/EEC, 2009/128/WE, OJEU).