Anopheles fluminensis Root is a member of the Arribalzagia Series in the subgenus Anopheles. We report the first record of this species in the department of Cochabamba, Bolivia. This species was sampled from two locations in the foothills of the eastern Andes Mountains within the Chapare Valley. Larvae were collected in fast-flowing, shaded streams at the edges of rocky pools. We provide the first sequence data for the rDNA of An. fluminensis, a partial sequence of the 5.8S and the internal transcribed spacer 2 (ITS2). The ITS2 of An. fluminensis, sequenced from two individuals at one site, was at least 596 bp, had 56.5% GC, and included three large repeats (approximately equal to 125 bp each). We describe a polymerase chain reaction protocol and species-specific primers for identifying this species in the Chapare Valley, Bolivia.