Resistance to the organophosphate insecticide chlorpyrifos was evaluated in females from six strains of Aedes aegypti (L.) that expressed high levels of cross-resistance to eight pyrethroid insecticides. Relative to LC₅₀ and LC₉₀ at 24 h of a susceptible New Orleans (NO) strain, three strains were highly resistant to chlorpyrifos (Coatzacoalcos, resistance ratio [RRLC₉₀] = 11.97; Pozarica, RRLC₉₀ = 12.98; and Cosoleacaque, RRLC₅₀ = 13.94 and RRLC₉₀ = 17.57), one strain was moderately resistant (Veracruz, RRLC₉₀ = 5.92), and two strains were susceptible (Tantoyuca and Martinez de la Torre, RRLC₅₀ and RRLC₉₀ < 5) in bottle bioassays according to Centers for Disease Control and Prevention. Furthermore, high levels of α- or β-esterase activity in the sample populations were correlated with resistance, suggesting that esterase activity may be a mechanism causing the development of organophosphate resistance in these populations. Overall, the populations in this study were less resistant to chlorpyrifos than to pyrethroids. Rotation of insecticides used in control activities is recommended to delay or minimize the occurrence of high levels of resistance to chlorpyrifos among local populations of Ae. aegypti. The diagnostic dose and diagnostic time for chlorpyrifos resistance monitoring was determined to be 85 µg per bottle and 30 min, respectively, using the susceptible NO strain.