Samples of stored maize from villages located in five different agroecological zones (southern lowlands, northern lowlands, Senqu river valley, foothills and mountains) of Lesotho were collected in 2009/10 and 2010/11 and assessed for contamination with toxigenic fungi. The water activity of all samples collected during the two seasons was <0.70. The total fungal populations of the maize from different regions in the two seasons was not significantly different (p > 0.05). Fusarium verticillioides, F. proliferatum and F. subglutinans predominated in different regions in both seasons based on molecular analyses. In the 2009/10 season, the isolates of these species all produced FB1, while in the 2010/11 season, very few produced FB1. A. flavus isolates (2009/10) were recovered from mountains and Senqu river valley samples while the 2010/11 isolates were predominantly from the foothills and northern lowlands. The mountain isolates of Aspergillus section Flavi produced the highest levels of AFB1 (20 mg kg(-1)). Aspergillus parasiticus was only isolated from the foothills, Senqu river valley and southern lowlands samples, and the AFB1 levels produced ranged from 'none detected' to 3.5 mg kg(-1). The Aspergillus ochraceous isolates were least frequently encountered in both seasons. In the 2009/10 season, the isolates from the northern lowlands produced ochratoxin A (OTA) in culture. No isolates of A. niger from different regions in both seasons produced any OTA. Multi-mycotoxin analyses of the maize samples were done for a range of mycotoxins. At least one sample from each region in both seasons was FB1-positive. FB1 levels for 2010/11 samples (7-936 μg kg(-1)) were higher than in the 2009/10 season (2-3 μg kg(-1)). In both seasons, the mountains registered the highest levels of FB1. Deoxynivalenol (DON) was recovered from all the samples analysed, with the highest mean contamination of 1,469 μg kg(-1) in samples from the northern lowlands. Moniliformin (MON) was detected from all agroecological zones in the two seasons (5-320 μg kg(-1) in 2009/10; 15-1,205 μg kg(-1) in 2010/11). Emerging toxins such as fusaproliferin (FUS) and beauvericin (BEA) were also detected. OTA was not detected in any of the samples analysed. Only one 2009/10 sample in the Senqu river valley was positive for AFB1. This is the first report on toxigenic fungi and multi-mycotoxin contamination of maize samples from subsistence farmers' stores in different agroecological zones of Lesotho.