Anopheles (Nyssorhynchus) marajoara Galvão & Damasceno (Diptera: Culicidae) is a member of the Albitarsis cryptic species complex and is a primary vector of human Plasmodium in parts of Brazil. We report the first record of An. marajoara in the department of Cochabamba and confirm its presence in the department of Santa Cruz, Bolivia. We also describe a PCR protocol producing a single amplicon (≈340 bp) of a section of the internal transcribed spacer 2 specific for members of the Albitarsis species complex; the polymerase chain reaction protocol did not amplify the DNA of 17 other species in the subgenus Nyssorhynchus. Digestion of the amplicon with the restriction endonuclease BfaI produces two fragments specific to An. marajoara in Bolivia.