A ribosomal DNA polymerase chain reaction technique (rDNA-PCR) that distinguishes the 5 more common and widespread members of the Anopheles gambiae complex failed to consistently identify specimens of Anopheles merus Dönitz collected in South Africa and Tanzania. When the original rDNA-PCR assay was applied to field-collected specimens or specimens from laboratory colonies established from these populations, bands diagnostic of both An. merus and An. quadriannulatus (Theobald) were amplified from all individual specimens. However, all the specimens tested had the polytene chromosome banding morphology or the superoxide dismutase isozyme that were diagnostic for An. merus. Replacement of the original An. quadriannulatus-specific primer with a new primer derived from another region of the rDNA intergenic spacer resulted in an alternative rDNA-PCR assay that accurately and consistently differentiated among specimens of An. merus, An. quadriannulatus, and An. arabiensis Patton. Anopheles gambiae Giles also may be distinguished by this assay if high percentage agarose gels or gels of other matrices with better resolving powers are used.