Resistance management for insecticide-treated nets (ITNs) remains a challenge. Options are limited, because a safe and highly active insecticide with a persistence of several months is required. These criteria have only been met by pyrethroids, although organophosphates (OPs) and carbamates have been considered as alternatives for impregnation of eave curtains. It has been observed that some pyrethroid-resistant mosquito strains show increased OP susceptibility over pyrethroid-susceptible strains (i.e., negative cross-resistance). The current study investigated whether this phenomenon applies to a range of mosquito species and strains, because a mixture or rotation strategy for resistance management could then be envisaged. Adult female mosquitoes from laboratory strains of Anopheles stephensi Liston, Anopheles gambiae Giles, and Culex quinquefasciatus Say were tested in World Health Organization susceptibility test kits. For An. stephensi, the highly pyrethroid-resistant DUB 234 strain showed the same level of resistance to malathion as the pyrethroid-susceptible DUB S. The malathion-resistant ST MAL strain was as susceptible to pyrethroids as the insecticide-susceptible BEECH. For An. gambiae, the malathion tolerance of the previously pyrethroid-resistant RSP strain was significantly higher than that of the insecticide-susceptible KWA. For Cx. quinquefasciatus, selection of the QUINQ strain with permethrin abolished preexisting resistance to the OP malathion as pyrethroid resistance increased, rendering the strain more susceptible to malathion than PEL SS. Some indication of negative cross-resistance to malathion was found for the permethrin-resistant MUHEZA strain. The occurrence of negative cross-resistance seems dependent on the history of insecticide selection and is not generally applicable. Resistance management for ITNs will need to use mechanisms other than negative cross-resistance to be effective.