The significant differences in mitochondrial genome size among seven races (B, E, M, T, U, W, and Y) of Podospora anserina have been found to be primarily due to the presence and/or absence of introns, including four introns not previously known to be optional. Information from physical mapping of races M and T, and sequence data from races A and s, was used to identify regions likely to contain insertions or deletions, which were then characterized using PCR and sequence analysis. Newly confirmed optional introns are the first intron of the large ribosomal RNA (LSUr1), the single intron of NADH dehydrogenase subunit 3 (ND3i1), the single intron in ATPase subunit 6 (ATPase6), and the fifth intron of cytochrome oxidase subunit I (COIi5). We have also found that race M exists in two forms as determined by mitochondrial DNA. These results bring to nine (including races A and s) the number of races characterized by mitochondrial intron content with a total of six known optional introns and one optional insertion. Eight of the nine races contain a distinct set of introns, providing a more reliable means for identification and comparison. The identification of optional mitochondrial introns in P. anserina may have evolutionary implications regarding the transfer and/or mobility of these introns.