Eukaryotic genes possess introns that must be spliced out following transcription. The origin of introns remains an unanswered question, one that is characterized by two different views: either they are ancient and generally undergoing loss or they are the result of more recent gain. We studied the intron organization of the fibrillarin gene from the sequenced genomes of twenty-five species representing all four eukaryotic kingdoms. Results showed that more complex, multicellular species had generally larger numbers and lengths of introns. Twenty-two different intron positions were used by the 127 introns found collectively in the fibrillarin genes from the twenty-five species. Several of these were shared in species across multiple kingdoms, a finding that was interpreted to indicate that these introns were present in early eukaryotic ancestors, as predicted by the introns-early model. In the ten deuterostome species examined, the fibrillarin introns showed a high level of evolutionary conservation, and the variations that did exist were best explained by four independent intron losses and no intron gain. The fungi as a group, however, showed much greater variation in the fibrillarin intron patterns, a result consistent with extensive intron loss, more recent intron gain, or a combination of both.