Genome size, life cycle, and ploidy were determined for Neottiella vivida (Nyl.) Dennis and N. rutilans (Fr.) Dennis. The relative DNA content was measured from fruit-bodies using cytofluorometry; genome size was obtained by comparison with two standards: conidia of Trichophea hemisphaerioides (23.3 Mb) and a sporeprint from Pleurotus ostreatus (25 Mb). Neottiella vivida and N. rutilans were found to have genomes of approximately 750 Mb and 530 Mb, respectively. The ploidy level of N. rutilans is 50, that of N. vivida was calculated to be 70. In N. vivida, meiotic division occurs in the ascus apex where giant mitochondria with a DNA content of 60 Mb, 54 Mb, and 30 Mb were found. The two species are morphologically very similar and can be distinguished only by their ascospore ornamentation, which is reticulated in N. rutilans and waited in N. vivida. Due to endoreduplication in the uninucleate ascospores of N. vivida, the value of their nuclear DNA content is 2C. In N. rutilans, endoreduplication is not arrested at the 2C value but may proceed at a different rate in spores. Thus N. rutilans reveals heterogeneity in ploidy levels of sporal nuclei. In N. vivida and N. rutilans, differences in spore ornamentation may result from different patterns of gene expression regulated by the ploidy-dependent gene.