By the first time an efficient methodology for micropropagation of poplar hybrids by temporary immersion in liquid medium was developed under photomixotrophic conditions. Influence of factors, such as plant regulator/balance in culture medium, number of explants or inoculums per bottle, immersion frequencies in Temporary Immersion Bioreactors (TIBs), reduced sucrose as priming treatment and photoperiod were studied in TIBs with 0.4 MPa CO2 at a light intensity of 100 M m−2 s−1. Protocol was standardized in three poplar clones recommended for their dendroenergetic traits. The highest multiplication rates were obtained by inoculating 20 and 24 nodal segments in TIBs with 200 ml of medium MS + 0.22 M TDZ. During winter (8 h photoperiod),the highest multiplication rates in TIBs were obtained with 8 h immersion frequencies, while in summer (14 h photoperiod) immersions every 6 h showed the best multiplication rates. The main percent of ex vitro rooted plants was recovered when plantlets were micropropagated in liquid medium supplemented with 1.5% sucrose. A drying pretreatment for 20 min followed by 10 min inoculation with amycorrhiza solution (Scleroderma spp.-Pisolithus tinctorius) allowed us to obtain 97.3% of poplars adapted to ex vitro environmental conditions. DNA patterns amplified with the ISSR primer (CTC)5 showed high genomic similarity without polymorphic bands between samples of the same hybrid, which indicated the non-occurrence of genomic changes in the studied populations. Overall, TIBs technology constitute an advance to the industrial development of clonal propagation of poplars for biomass production, in comparison with the classical micropropagation.