Asparagus spears collected from a total of six commercial plantings in Austria during the main harvest periods in May and June of 2003 and 2004 were examined for endophytic colonization by Fusarium spp., particularly F. proliferatum. Potentially toxigenic fungi such as F. proliferatum were isolated and identified by morphological characteristics using light microscopy. Fumonisin B1 in F. proliferatum-infected asparagus spears was detected with IAS-HPLC-FLD or HPLC-MS/MS. The identity of endophytic fungi colonizing of a total of 816 individual spears was determined. The incidence of infection by F. proliferatum and other Fusarium spp. was highly dependent on location and sampling date. The dominant Fusarium species among the endophytic microflora was F. oxysporum. Other frequently isolated species included F. proliferatum, F. sambucinum, F. culmorum, F. avenaceum and F. equiseti. The incidence of F. proliferatum-infected asparagus spears was less than 10% at four of the six sampling locations. At the two remaining locations, 20–47% of the spears examined were infected with F. proliferatum. Further exploration of FB1 generation in asparagus is required because the low levels of FB1 (10–50 (μ;g/kg) detected in harvested spears in 2003 and 2004 cannot be explained by the results of this study.