Females of the genusMansoniafeed on the blood of humans, livestock, and other vertebrates to develop their eggs. The females’ biting behavior may cause severe disturbance to blood hosts, with a negative impact on public health and economics. Certain species have been identified as potential or effective disease vectors. The accurate species identification of field-collected specimens is of paramount importance for the success of monitoring and control strategies.Mansonia(Mansonia) morphological species boundaries are blurred by patterns of intraspecific heteromorphism and interspecific isomorphism. DNA barcodes can help to solve taxonomic controversies, especially if combined with other molecular tools. We used cytochrome c oxidase subunit I (COI) gene 5′ end (DNA barcode) sequences to identify 327 field-collected specimens ofMansonia(Mansonia) spp. The sampling encompassed males and females collected from three Brazilian regions and previously assigned to species based on their morphological characteristics. Eleven GenBank and BOLD sequences were added to the DNA barcode analyses. Initial morphospecies assignments were mostly corroborated by the results of five clustering methods based on Kimura two-parameter distance and maximum likelihood phylogeny. Five to eight molecular operational taxonomic units may represent taxonomically unknown species. The first DNA barcode records forMansonia fonsecai,Mansonia iguassuensis, andMansonia pseudotitillansare presented.