The aim of the study was to determine the level of aflatoxin B1 (AFB1) in maize sampled from farms and feed factories situated in Northern, Central and Eastern Croatia during 2013, following the occurrence of cow milk AFM1 contamination. Maize samples (n = 633) were analysed using Enzyme-Linked Immunosorbent Assay (ELISA) as a screening method and High Performance Liquid Chromatography Tandem Mass Spectrometry (LC–MS/MS) as a confirmatory method. Mean AFB1 value found in maize coming from all investigated regions equalled to 81 μg/kg, with the maximal value of 2072 μg/kg found in maize obtained from Eastern Croatia. The observed contamination might have arisen on the grounds of extremely hot (>98%) and dry (<2%) weather witnessed from May to September 2012 during the maize growth and harvesting period, which might have favoured AFB1 production and consequently the contamination of dairy cattle feeds. In order to prevent the adverse effects of AFB1 on humans and animals, and also to reduce losses in agricultural production, systematic monitoring and further investigations of AFB1 contamination are necessary. •Aflatoxin B1 in maize from farms and feed factories in Croatia during 2013 was determined.•ELISA and LC–MS/MS methods were implemented.•Very high levels of AFB1 were observed in maize from Eastern Croatia.•Hot and dry weather witnessed in 2012 might have favoured AFB1 production.•Systematic monitoring and further investigations of AFB1 contamination are necessary.