An analysis of the ovarian polytene chromosomes of Anopheles cruzii from three localities in Southeast Brazil revealed the existence of two genetic entities within this morphologically uniform taxon. These cryptic species differed in the banding patterns of the X chromosome and 3L arm. A pattern of bands that cannot be explained by the fixation of any of the known inversions in chromosome X was revealed and named chromosomal form B to distinguish it from the standard pattern of this X chromosome, form A. Each chromosomal form is characterized by a different set of inversions. The lack of heterozygotes (A/B) for these X chromosome forms in populations where both forms coexist is evidence of absence or limited gene flow between the two groups. Key words: Anopheles cruzii, inversion polymorphism, sibling species.