Fumonisins are mycotoxins produced primarily by Fusarium verticillioides and F. proliferatum on maize. These mycotoxins are secondary, carcinogenic metabolites with a worldwide distribution. This study was performed to evaluate the relationship between weather variables, the colonization of grain by F. verticillioides and resultant fumonisin contamination. Grain colonization by Fusarium spp. was determined using quantitative real-time PCR (qPCR) and contamination with fumonisins using HPLC analysis. Results indicated high natural infection by fumonisin-producing Fusarium spp. and fumonisin concentrations in warmer production areas such as Northern Cape, North West and Free State Provinces. Site-specific weather data, including temperature, radiation, humidity, rainfall and evapo-transpiration were provided by the ARC-Institute for Soil Water and Climate's meteorology office. Stepwise multiple regression analysis selected the variables mean maximum temperature and mean minimum humidity (days 1–14 post-silking) as having significant relationships with colonization of maize kernels by fumonisin-producing Fusarium spp.. Further relationships were calculated using the non-linear, 3-dimensional Lorentzian equation (Sigmaplot 10.0). The optimum temperature for colonization of maize by Fusarium spp. were 28.97, 32.14 and 30.40 °C and the optimum minimum humidities were calculated at 27.29, 31.86 and 29.74% respectively over the three recorded seasons. The application of correlation analysis and stepdown multiple regression analysis on sequential means of weather data and the inclusion of observed fungal biomass as a variable, suggested two phases in the development of fumonisins in maize kernels i.e. colonization of maize tissues during the early post-silking stage, followed by fumonisin production during the dough stage of grain fill. Based on this statistical model it would appear that fumonisin production by a specific biomass of fumonisin producing Fusarium spp. on grain was influenced largely by temperature and less so by rainfall. Optimum fumonisin concentrations were obtained with fungal biomasses of 39.94 pg during 2007, 47.65 pg during 2008 and 90.69 pg during 2009. Optimum temperatures for fumonisin production were 30.33, 31.12 and 29.80 °C for 2007–2009, respectively. Although these statistical models were not consistent over seasons with regard to actual prediction values, they were consistent regarding time of fungal infection and fumonisin production. It is evident that additional driving variables need to be identified. •The effect of prevailing weather conditions on F. verticillioides colonization and fumonisin contamination was evaluated.•High natural fungal colonization and corresponding fumonisin contamination occurred in warmer maize production areas.•Mean maximum temperature and mean minimum humidity had significant relationships with fungal colonization of maize grain.•The statistical model suggests fumonisin production in maize kernels early post-silking and at dough stage of grain fill.