Sexual isolation inDrosophila is typically measured by multiple-choice mating tests. While many environmental variables during such tests are controlled by the researcher, there are some factors that are usually uncontrolled. We demonstrate, usingDrosophila melanogaster andD. pseudoobscura flies, that the temperature of rearing, preadult density, and level of consanguinity, can all produce differences in mating propensity between genetically equivalent flies. These differences in mating propensity, in turn, can give rise to statistically significant results in multiple-choice mating tests, leading to positive isolation values and the artifactual inference of sexual isolation between populations. This fact agrees with a nonrandom excess of significant positive tests found in a review of the literature ofDrosophila intraspecific mating choice. An overestimate of true cases of sexual isolation inDrosophila in the literature can, therefore, not be ruled out.