Plants’ genetic improvement continues to be crucial for modern agriculture, while biotechnology can offer efficient tools that enhance the selection and recommendation processes of elite clones. This work established a suitable methodology for the regeneration of blueberry (Vaccinium corymbsum) plants in cultures with colchicine. This could be considered a basis for producing populations for the selection of clones following a genetic improvement program assisted by biotechnology. The factors studied were: (a) explant type (leaf discs; nodal segments); (b) colchicine concentration (0, 0.5, 1, and 2 mg/L); and (c) time of exposure to colchicine (1, 2, 3, 5, and 30 days). The basal medium McCown’s Woody Plant (WP) supplemented with 2 mg/L 2iP and 1 mg/L BAP was used with the commercial genotype Duke as a model. A total of 1957 blueberry clones were produced in a medium with 1 mg/L colchicine, distributed at different exposure times. Flow cytometry analyses revealed the following patterns: single patterns for random samples of control plants (Duke donor) and some clones regenerated on colchicine; double patterns for chlorotic plants regenerated on colchicine. Triple and quadruple patterns were observed in callus tissues that did not regenerate plants on colchicine. Populations of plants regenerated in colchicine (6787) and control plants regenerated in in vitro culture without colchicine were adapted under greenhouse conditions. The variables evaluated at this stage were adaptability, height, diameter, number of leaves, incidence of diseases, flowering capacity, and agrobotanical traits. Selected clones demonstrating phenotypic variability (157 clones) were transplanted to field conditions. From the clonal field trial conducted under minimum tillage conditions, 38 clones were selected for improved traits related to the agricultural yield and nutritional quality of the fruits. Of these, six clones showed the highest agronomic performance and adaptability to adverse environmental conditions compared to the Duke donor genotype. It is recommended that these clones continue genotype × environment interaction trials at different locations.