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13 There is increasing interest in comparing species of related organisms for their susceptibility 14 to thermal extremes in order to evaluate potential vulnerability to climate change. 15 Comparisons are typically undertaken on individuals collected from the field with or without 16 a period of acclimation. However this approach does not allow the potential contributions of 17 environmental and carry-over effects across generations to be separated from inherent species 18 differences in susceptibility. To assess the importance of these different sources of variation, 19 we here consider heat and cold resistance in Drosophilid species from tropical and temperate 20 sites in the field and across two laboratory generations. Resistance in field-collected 21 individuals tended to be lower when compared to F1 and F2 laboratory generations, and 22 species differences in field flies were only weakly correlated to differences established under 23 controlled rearing conditions, unlike in F1 F2 comparisons. This reflected large 24 environmental effects on resistance associated with different sites and conditions experienced 25 within sites. For the cold recovery assay (after 8 h cold stress) there was no strong evidence 26 of carry-over effects, whereas for the heat knockdown (heat) and cold recovery assays (after 27 2 h cold stress) there was some evidence for such effects. However for heat these were 28 species-specific in direction. Variance components for inherent species differences were 29 substantial for resistance to heat and 8 h cold stress, but small for 2 h cold stress, though this 30 may be a reflection of the species being considered in the comparisons. These findings 31 highlight that inherent differences among species are difficult to characterize accurately 32 without controlling for environmental sources of variation and carry-over effects. Moreover 33 they also emphasize the complex nature of carry-over effects that depend on the nature of 34 stress traits and the species being evaluated. 35