We analyzed nuclear (second internal transcribed spacer and white gene) and mitochondrial (cytochrome c oxidase subunit I) data from Anopheles konderi collected in the Amazonian states of Acre, Amapá, and Rondônia and the southern Brazilian state of Paraná. This was the first record of An. konderi in the state of Paraná. We found a high degree of genetic divergence within the Amazonian region and support for An. konderi as a species complex, possibly consisting of 3 species.