Chagas disease remains a major public health problem in Latin America, with Triatoma dimidiata (Latreille, 1811) recognized as one of the principal vectors in Mexico. The prolonged use of pyrethroids for vector control has raised concerns about the emergence of resistance, often associated with knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC). Monitoring insecticide susceptibility and identifying associated resistance mechanisms are, therefore, critical for effective surveillance and control. In this study, we evaluated the susceptibility of T. dimidiata from Yucatan, Mexico, to deltamethrin and permethrin using topical application bioassays and sequenced VGSC domain II to detect potential kdr substitutions. We report, for the first time in T. dimidiata, the presence of the L1014F mutation in a population exhibiting resistance to permethrin (RR50 = 61) and susceptibility to deltamethrin (RR50 = 0.17). Although the functional impact of this mutation in T. dimidiata remains unknown, its detection provides new information on the molecular variability of this vector. These findings highlight the need to integrate kdr mutation screening into insecticide resistance surveillance programs.