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Spinosad has been used to control Tuta absoluta in Brazil for more than a decade but will eventually be replaced by spinetoram despite the risk of cross-resistance. Therefore, the susceptibility to both molecules and the activity of detoxification enzymes were determined for eight representative populations of T. absoluta to assess resistance and the risk of cross-resistance. The LC50 values for spinosad varied from 0.007 (Pelotas) to 0.626 mg/L (Sumare); the LC50 values for spinetoram varied from 0.047 (Pelotas) to 0.308 mg/L (Sumare). The LC99 values for spinosad varied from 0.23 (Pelotas) to 11.56 mg/L (Venda Nova do Imigrante); the LC99 values for spinetoram varied from 0.55 (Pelotas) to 6.71 mg/L (Iraquara). The resistance levels ranged from 1.0- to 93.8-fold (RR50) and 1.0- to 51.5-fold (RR99) for spinosad and from 1.0- to 6.5-fold (RR50) and 1.0- to 12.1-fold (RR99) for spinetoram. The concentration-mortality responses to spinetoram were more homogeneous than those to spinosad. A strong correlation between the susceptibilities of T. absoluta populations to spinosad and spinetoram was observed, showing the similarity of the mode of action of both molecules and producing cross-resistance between them. The β–esterase activity of T. absoluta populations was correlated with spinosyn susceptibility, suggesting a potential contribution of the enzyme to evolved spinosyn resistance. The evolution of resistance to spinosyns in T. absoluta observed in this study suggests that strategies to mitigate resistance must be carefully implemented over the short term and that rotation with other products is encouraged.