Dengue virus (DENV) is the etiological agent of dengue fever (DF), which is among the most prevalent vector-borne diseases in the tropics. In 2022, the Colombian health surveillance system reported more than 69,000 cases of DF. As part of a hospital-based fever surveillance study, acute-phase sera were collected from 4,545 patients with suspected dengue between 2020 and 2023 in three municipalities of Colombia. Combined reverse transcription-polymerase chain reaction and antigen rapid testing confirmed that 376 patients (8.3%) had DF. The virus was isolated in cell culture from 166 of these patients (44.1%), and genome sequencing was performed successfully on 122 (73.5%). Three DENV serotypes (1, 2, and 3) were identified. Phylogenetic analyses of the DENV-2 sequences revealed that 42 of 50 of the isolates (84%) belonged to the DENV-2 cosmopolitan genotype lineage, clustering with sequences from Asia, Peru, and Brazil. We report the detection, isolation, and whole-genome sequencing (11 Kb) of the DENV-2 cosmopolitan genotype and its recent introduction to Colombia.