The sequence of a 2.3 kb long DNA segment derived from the 5'-most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako.