Background <p><i>Aedes aegypti</i> chemical control remains the major strategy to prevent dengue, Zika, and Chikungunya outbreaks. The Cuba archipelago organizes a constant surveillance in its entire territory to follow up the distribution and infestation levels, but also insecticide resistance of <i>Ae. aegypti</i>, to estimate arbovirus transmission risk for its inhabitants. The objective of this study was to determine temephos susceptibility status in the <i>Ae. aegypti</i> population from western, central, and eastern regions of Cuba.</p> Methods <p><i>Aedes</i> larvae bioassays were performed following World Healthy Organization (WHO) methodology. Entomological samples were collected in the western (Pinar del Rio and Matanzas), central (Santa Clara [Villa Clara], Cienfuegos, Sancti Spíritus, and Camagüey), and eastern (Las Tunas, Holguín, Santiago de Cuba, and Guantánamo) regions of Cuba from February 2022 to June 2023.</p> Results <p>This study showed susceptibility in Las Tunas population (RR₅₀ = 0.94). Santa Clara [Villa Clara] (RR₅₀ = 8.94), Cienfuegos (RR₅₀ = 5.88), and Sancti Spíritus (RR₅₀ = 6.7) populations showed moderate resistance and the rest showed high resistance (RR₅₀ &gt; 10) to temephos.</p> Conclusions <p>Most <i>Ae. aegypti</i> populations tested from western, central and eastern regions of Cuba showed spatial homogeneity of temephos resistance owing to the intensive use of this larvicide since 1981. Insecticide resistance management by the National Vector Control Program is required to reverse temephos resistance development.</p> Graphical abstract <p></p>