The Drosophila melanogaster gene Dscam1 potentially generates 38,016 distinct isoforms via mutually exclusive splicing, which are required for both nervous and immune functions. However, the mechanism underlying splicing regulation remains obscure. Here we show apparent evolutionary signatures characteristic of competing RNA secondary structures in exon clusters 6 and 9 of Dscam1 in the two midge species (Belgica antarctica and Clunio marinus). Surprisingly, midge Dscam1 encodes only ∼6000 different isoforms through mutually exclusive splicing. Strikingly, the docking site of the exon 6 cluster is conserved in almost all insects and crustaceans but is specific in the midge; however, the docking site-selector base-pairings are conserved. Moreover, the docking site is complementary to all predicted selector sequences downstream from every variable exon 9 of the midge Dscam1, which is in accordance with the broad spectrum of their isoform expression. This suggests that these cis-elements mainly function through the formation of long-range base-pairings. This study provides a vital insight into the evolution and mechanism of Dscam1 alternative splicing.