Microdochium nivale and Microdochium majus are two of fungal species found in the Fusarium Head Blight (FHB) complex infecting small grain cereals. Quantitative real-time PCR assays were designed to separate the two Microdochium species based on the translation elongation factor 1a gene (TEF-1a) and used to analyse a total of 374 seed samples of wheat, barley, triticale, rye and oat sampled from farmers’ fields across Denmark from 2003 to 2007. Both fungal species were detected in the five cereal species but M. majus showed a higher prevalence compared to M. nivale in most years in all cereal species except rye, in which M. nivale represented a larger proportion of the biomass and was more prevalent than M. majus in some samples. Historical samples of wheat and barley from 1957 to 2000 similarly showed a strong prevalence of M. majus over M. nivale indicating that M. majus has been the main prevalent Microdochium species in Danish cereals for at least 50 years. PCA analysis of the two quantified Microdochium species in wheat, barley and triticale samples generally showed co-existence of M. majus and M. nivale in all three cereal species. Strobilurin resistance in M. nivale/majus was analysed in selected wheat samples from 2003 to 2007, selected barley samples from 2007 as well as in historical samples from 1957 to 2000 using CAPS analysis to detect the G143A substitution. The results confirm strobilurin resistance from 2003 in the Microdochium populations of wheat and also confirmed resistance in barley for the first time. The presence of strobilurin resistance should be considered in future fungicide control strategies.