Introns are usually non-coding sequences interrupting open reading frames in pre-mRNAs [D1,2]. Stwintrons are nested spliceosomal introns, where an internal intron splits a second donor sequence into two consecutive splicing reactions leading to mature mRNA. In Hypoxylon sp. CO27-5, 36 highly sequence-similar [D1,2] stwintrons are extant (sister stwintrons). An additional 81 [D1,2] sequence-unrelated stwintrons are described here. Most of them are located at conserved gene positions rooted deep in the Hypoxylaceae. Absence of exonic sequence bias at the exon–stwintron junctions and a very similar phase distribution were noted for both groups. The presence of an underlying sequence symmetry in all 117 stwintrons was striking. This symmetry, more pronounced near the termini of most of the full-length sister stwintrons, may lead to a secondary structure that brings into close proximity the most distal splice sites, the donor of the internal and the acceptor of the external intron. The Hypoxylon stwintrons were overwhelmingly excised by consecutive splicing reactions precisely removing the whole intervening sequence, whereas one excision involving the distal splice sites led to a frameshift. Alternative (mis)splicing took place for both sister and uniquely occurring stwintrons. The extraordinary symmetry of the sister stwintrons thus seems dispensable for the infrequent, direct utilisation of the distal splice sites.