It is essential to assess the environmental risk that Bt maize may hold and to study its effect on species assemblages that fulfil a variety of ecosystem functions. Environmental risk assessment can be improved through the use of an ecological model which can be applied to a specific environment, so that local species can be classified functionally and prioritized to identify potential test species. Although the stem borers, Busseola fusca (Lepidoptera: Noctuidae), and Chilo partellus (Lepidoptera: Crambidae), are the target species of Bt maize in South Africa various other Lepidoptera species are also directly exposed to Bt toxin. In this paper an ecological approach was followed for selection of non-target Lepidoptera species for ecological risk assessment of Bt maize, using data collected over a two-year period on Lepidoptera biodiversity on maize. Nine primary non-target lepidopterous consumers were identified. A selection matrix was developed in which each species was ranked for its maximum potential exposure to Bt toxin by assessing its occurrence, abundance, presence and linkage to other host plants in the maize ecosystem. Non-target Lepidoptera most likely to be affected were identified and prioritized for future testing and inclusion in risk assessments. Several non-target species were prioritized for their close association with maize, general occurrence in the maize-growing regions and their potential for economic damage should they become secondary pests. Through use of the selection matrix, knowledge gaps were identified for future research and guidance for the design of ecologically realistic experiments. Non-target species populations with the highest maximum potential exposure to Bt toxin are Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae), Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) and Acantholeucania loreyi (Duponchel) (Lepidoptera: Noctuidae). Eublemma gayneri (Rothschild) (Lepidoptera: Noctuidae) can be considered to be a ‘value unknown’ species in the wild. Because of their sporadic occurrence, Agrotis segetum (Denis & Schiffermüller) (Lepidoptera: Noctuidae) and Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) are considered to be of lesser importance than H. armigera and A. loreyi, but should also be considered during pre-and post-release monitoring.