Microdochium nivale and M. majus are fungal plant pathogens that cause cool-temperature diseases on grasses and cereals. Nucleotide sequences of four genetic regions were compared between isolates of M. nivale and M. majus from Triticum aestivum (wheat) collected in North America and Europe and for isolates of M. nivale from turfgrasses from both continents. Draft genome sequences were assembled for two isolates of M. majus and two of M. nivale from wheat and one from turfgrass. Dendograms constructed from these data resolved isolates of M. majus into separate clades by geographic origin. Among M. nivale, isolates were instead resolved by host plant species. Amplification of repetitive regions of DNA from M. nivale isolates collected from two proximate locations across three years grouped isolates by year, rather than by location. The mating-type (MAT1) and associated flanking genes of Microdochium were identified using the genome sequencing data to investigate the potential for these pathogens to produce ascospores. In all of the Microdochium genomes, and in all isolates assessed by PCR, only the MAT1-2-1 gene was identified. However, unpaired, single-conidium-derived colonies of M. majus produced fertile perithecia in the lab. This finding contrasts with the cannonical requirements for sexual spore production among the Ascomycota. To further explore this, MAT1 and flanking gene sequences were identified in the genome sequences of six additional species from Xylariaceae, no homologs of known MAT1-1-1 genes were detected, suggesting that the control of sexual reproduction among the Xylariaceae may be differentally regulated relative to other Sordariomycete species. Detached leaves of T. aestivum and Poa pratensis (Kentucky bluegrass) were inoculated with either M. nivale or M. majus and were incubated at either 23 ºC or at 4 ºC to investigate the infection processes of these pathogens. Despite reported field host preferences, the two pathogens were equally virulent on both host plants at the temperatures investigated. The results presented here reveal genetic, but not pathogenic, differences between M. nivale and M. majus and further demonstrate that sub-populations may exist within the groups of these pathogens on different host plants.