Aedes (Stegomyia) albopictus has been identified for the first time in the municipality of La Tebaida (Quindio department of Colombia), an area with active transmission of dengue fever by Ae. (Stegomyia) aegypti. Specimens of Ae. albopictus were detected in a tire used as an ovitrap in a remnant of bamboo plantation in a rural area of the municipality of La Tebaida; Ae. aegypti presented co-occurrence and both species were molecularly typed using cytochrome oxidase I (DNA barcode region). The first DNA barcode sequences were recorded for 13 Colombian specimens of Ae. albopictus, identifying an asiatic origin (Singapore) and demonstrating the utility of this method for molecular identification. These sequences can be used to identify genetic flow with other populations in Colombia, in ecological studies, and in studies of vector incrimination in outbreaks of emerging and re-emerging arbovirus in Colombia.