In March 2013 a large shipment of maize, intended for feed was subject of an alert in the Rapid Alert System for Food and Feed of the European Commission (EC) because the aflatoxin B1 (AFB1) level in the load exceeded the EC regulated maximum level of 20 µg/kg. Since the shipment had passed import controls and was already distributed (mainly to German farms), a massive recall followed. The aim of the current study was to investigate questions, raised by authorities and industry, related to the effectivity of EU sampling procedures, the influence of sample homogenisation procedures and sample storage conditions on the test results, and fungal identification as unexpected mycotoxins were identified during this study. The Netherlands Food and Consumer Product Safety Authority seized a shipload of maize in July 2013, suspected to be contaminated with AFB1. The shipload was sampled according to the 2009 and 2013 EC Sampling Regulations to compare the outcomes of both sampling protocols. Mycotoxin analysis of the incremental samples showed high mean levels of AFB1, aflatoxin G1 (AFG1), and ochratoxin A (OTA). Also an extreme inhomogeneous distribution of aflatoxins and OTA was proven. Analysis of samples homogenised according to the slurry method showed improved performance as compared to samples homogenised through dry homogenisation. Sampling and sample homogenisation according to the Regulation from 2013 showed a closer estimate of the ‘true’ AFB1 content as compared to sampling according to the Regulation from 2009. No influence of laboratory storage conditions on AFB1 concentration could be determined. Fungal identification revealed Aspergillus flavus as the main source of AFB1 in this shipment. Infrequent occurrence of Aspergillus parasiticus might have been the source of AFG1. The occurrence of sometimes large amounts of OTA could not be explained, however it was suggested that Aspergillus welwitschiae might have played a role.