The effect on spatial variability of leaf temperature of inoculating grapevine leaves (Vitis vinifera L. cv. Riesling) with a fungal pathogen (Plasmopara viticola) was studied in either well irrigated or non-irrigated plants. The results from thermal imagery were compared with stomatal conductance measured by leaf gas exchange. The high sensitivity of leaf temperature to stomatal conductance means that infrared thermography can be used to monitor irregularities in temperature at an early stage of development after either infection or other stress-related changes affecting the amount of water transpired. Contrasting thermal effects due to the pathogen attack were found between measurements on well irrigated and water stressed plants. With irrigated vines, pathogen development caused an increase in leaf temperature at the point of infection. In contrast, under severe water stress, the inoculated plants showed a lower temperature at the sites of inoculation compared with the rest of the leaf. Analysis of the spatial and temporal sensitivity of the temperature profile, obtained from the deviation of individual pixels from the mean along a straight line, successfully distinguished between healthy and infected positions on the leaf irrespective of the plant water status. Under greenhouse conditions and for predefined areas of the leaf surface, evidence was also acquired for characteristic thermal responses to be apparent not later than 4 days past inoculation; that is, at least 3 days before visible symptoms appeared. Thus, early and remote detection using thermal imagery has the potential for pre-symptomatic diagnosis of biotic stress.