The ascomycetous fungus Cryptendoxyla hypophloia contains an insertion of 433 base pairs in the genes encoding nuclear small subunit ribosomal RNA. Secondary structure analyses of the insert reveal characteristics indicative of a Group I intron, including elements P, Q, R, and S; however, the sequences of these conserved regions deviate significantly from recognized consensus sequences for Group I introns. Principal-components analysis, based on 79 nucleotide positions from the conserved core sequences of 93 Group I introns, identified 17 introns similar to that of C. hypophloia. This grouping, which includes inserts from phylogenetically diverse organisms, cannot readily be classified in any previously recognized major group of Group I introns. We propose the creation of a new group, IE, to accommodate these sequences, and discuss the evolutionary relationships between group IE and other major groups of Group I introns.