Neonicotinoid insecticides are environmentally persistent and highly water-soluble, and thus are prone to leaching into surface waters where they may negatively affect non-target aquatic insects. Most of the research to date has focused on imidacloprid, and few data are available regarding the effects of other neonicotinoids or their proposed replacements (butenolide insecticides). The objective of this study was to assess the toxicity of six neonicotinoids (imidacloprid, thiamethoxam, acetamiprid, clothianidin, thiacloprid, and dinotefuran) and one butenolide (flupyradifurone) to Hexagenia spp. (mayfly larvae). Acute (96-hour), water-only tests were conducted, and survival and behaviour (number of surviving mayflies inhabiting artificial burrows) were assessed. Acute sublethal tests were also conducted with imidacloprid, acetamiprid, and thiacloprid, and in addition to survival and behaviour, mobility (ability to burrow into sediment) and recovery (survival and growth following 21 day in clean sediment) were measured. Imidacloprid concentrations measured in North American surface waters have been found to meet or exceed those causing toxicity to Hexagenia, indicating that environmental concentrations may adversely affect Hexagenia and similarly sensitive non-target aquatic species.