A new procedure for blueberry (Vaccinium corymbosum L.) micropropagation in programmed Temporary Immersion Bioreactors (TIBs based on two separate bottles) was developed for the commercial genotypes Biloxi, Sharp Blue and Brillita. Plant cultures were developed in a controlled environment with 0.4 MPa CO2 enrichment, sucrose-reduced medium, and light intensity of 60 M m−2 ·s−1. Principal component analysis showed that component 1 (C1) grouped 64.08% of the total variability, while the first two components accounted for 86.97%. Representation of the principal components demonstrated three clusters corresponding with the blueberry genotypes, and within each cluster plants micropropagated in agar-base medium grouped separately from those plants multiplied in TIBs. Both plant number and total internodes traits (related to the productive efficiency) were demonstrated superior in blueberries propagated in TIBs. Additionally, when transferred to greenhouse conditions, blueberries propagated in TIBs showed higher adaptability and growing rates than those cultured by the conventional approach, altogether evidencing the occurrence of a photomixotrophic stage in the vitroplantlets cultured in TIBs.