Background: Aedes aegypti is a vector of high relevance, since it transmits several arboviruses, including dengue, chikungunya and Zika. Studies on vector biology are usually conducted with laboratory strains presenting a divergent genetic composition from feld populations. This may impair vector control policies that were based on laboratory observations employing only long maintained laboratory strains. In the present study we characterized a laboratory strain interbreed with Ae. aegypti collected from fve diferent localities in Rio de Janeiro (Aedes Rio), for insecticide resistance (IR), IR mechanisms, ftness and Zika virus infection. Methods: We compared the recently established Aedes Rio with the laboratory reference strain Rockefeller. Insecticide resistance (deltamethrin, malathion and temephos), activity of metabolic resistance enzymes and kdr mutation frequency were determined. Some life table parameters (longevity, blood-feeding, number and egg viability) and Zika virus susceptibility was also determined. Results: Aedes Rio showed resistance to deltamethrin (resistance ratio, RR50=32.6) and temephos (RR50=7.0) and elevated activity of glutathione S-transferase (GST) and esterases (α-EST and pNPA-EST), but not acetylcholinesterase (AChE). In total, 92.1% of males genotyped for kdr presented a “resistant” genotype. Weekly blood-fed females from both strains, presented reduced mortality compared to sucrose-fed mosquitoes; however, Aedes Rio blood-fed females did not live as long (mean lifespan: Rockefeller=70±3.07; Aedes Rio=53.5±2.16 days). There were no diferences between strains in relation to blood-feeding and number of eggs, but Aedes Rio eggs presented reduced viability (mean hatch: Rockefeller=77.79±1.4%; Aedes Rio=58.57±1.77%). Zika virus infection (plaque-forming unit, PFU) was similar in both strains (mean PFU±SE: Aedes Rio: 4.53×104±1.14×104 PFU; Rockefeller: 2.02×104±0.71×104 PFU). Conclusion: Selected conditions in the feld, such as IR mechanisms, may result in pleiotropic efects that interfere in general physiology of the insect. Therefore, it is important to well characterize feld populations to be tested in parallel with laboratory reference strains. This practice would improve the signifcance of laboratory tests for vector control methods.