Fusarium causes significant post-harvest quality losses and mycotoxin contamination in stored wheat but the colonisation dynamics of the grain and how this may be affected by the initial inoculum position in the grain mass is poorly understood. This study examined the 3D growth kinetics and mycotoxin production (deoxynivalenol and zearalenone) by F. graminearum during hyphal colonisation from different initial inoculum positions in wheat microcosms (top-centre, bottom-centre, and bottom-side) maintained at two water activities (aw; 0.95 and 0.97). Clear jars were used to visually follow the colonisation dynamics. Fungal respiration and associated dry matter loss (DML) and ergosterol were also quantified. Colonisation dynamics was shown to be affected by the inoculation position. At the end of the colonisation process, fungal respiration and DML were driven by the inoculation position, and the latter also by the prevailing aw. Fungal biomass (ergosterol) was mainly affected by the aw. The initial inoculum position did not affect the relative mycotoxin production. There was a positive correlation between respiration and ergosterol, and between mycotoxin production and colonisation indicators. We suggest that spatially explicit predictive models can be used to better understand the colonisation patterns and mycotoxin contamination of stored cereal commodities and to aid more effective post-harvest management.