Mosquitoes of the Anopheles bancroftii group collected from Northern Australia and Papua New Guinea (PNG) were investigated for sequence variation within the ribosomal DNA ITS2. Wing fringe morphology originally used to identify members of this group was compared to genotypes identified by restriction fragment length polymorphism analysis (RFLP) and heteroduplex analysis (HDA) of the rDNA ITS2. Members of this group separated into four RFLP genotypes (A, B, C and D) with some genotypes displaying wing fringe polymorphisms. Heteroduplex analysis of the ITS2 within and between populations identified genotype A as containing two geographically separate ITS2 sequences: A1 from the Northern Territory of Australia and A2 from Queensland and the Western Province of PNG. Genotypes B and C and genotypes C and D were found sympatric and appeared to be evolving independently suggesting the possibility of cryptic species. Genotype C contained two ITS2 sequence types within the genome.