AbstractThe primary and secondary structure of the internal transcribed spacers of ribosomal DNA (ITS1 and ITS2) and their utility for phylogenetic analysis of closely related species were examined using theAnopheles gambiaecomplex as a model. Restriction mapping revealed an unusual architectural feature in the ITS1 of several members of anAn. gambiaecryptic species complex. Multiple spacer lengths are prevalent inAn. merusandAn. melasand are due to variable numbers of a repeated 250 bp sequence. Secondary structure analysis indicated that the repeat forms a helix and loop that may be involved in rDNA processing. Intra‐and interspecific polymorphism within the species complex were further examined by DNA sequencing of forty‐eight ITS2 clones obtained by polymerase chain reaction from individuals of the five species. Interspecies variation in the approximately 426 bp ITS2 sequence ranged between 0.4% and 1.6%; intraspecies variation ranged from 0.07% inAn. arabiensisto 0.43% inAn. gambiae.lntraindividual variation ranged from 0% in four individuals to a high of 0.4% in oneAn. quadriannulatusspecimen. None of the variants were shared between species. The low level of variation supports the hypothesis that species of the complex evolved recen