Polymorphisms for the presence or absence of supernumerary bands, for band size, nucleolar expression and the location of secondary nucleolar organisers found in the polytene chromosomes of Simulium ornatipes are described. In all the seven band polymorphisms analysed, six of which involve single bands and one a multiple band complex (IILH), phylogenetic evidence shows that there has been an addition of material. IILH consists of seven amplified C-bands, two supernumerary C-bands and a supernumerary segment involving two C-bands and an interband region. These bands are linked to the peracentric inversion IIL-3, five of the bands being located only within inverted segments. Comparison with mitotic chromosomes suggests that IILH heterochromatin is not under replicated in polytene chromosomes. Recombination between IILH components occurs at a very low level which is insufficient to disrupt the integrity of the polymorphism. It is concluded therefore that the complex evolved in a sequential series, the origin of IIL-3 being the first step. Single band polymorphisms, some of which are also linked to inversions, show similar heterochromatic properties to the IILH bands. A mechanism of selective DNA sequence amplification is proposed to explain increase in band size and the accompanying heterochromatinization. Most supernumerary bands may be amplifications of submicroscopic bands. Nucleolar organisers show heteromorphism for expression and rare secondary nucleoli are found on all chromosomes. It is argued that a multiplicity of sites for ribosomal genes are distributed in the genome and that selective sequence amplification, similar to that proposed above, can increase these sequences to a functional level at any of the sites. This would explain the lability of nucleolar sites in different blackfly species.