In this study we examine the hypothesis that divergent natural selection produces genetic differentiation among populations in plant defensive strategies (tolerance and resistance) generating adaptive variation in defensive traits against herbivory. Controlled genetic material (paternal half-sib families) from two populations of the annual Datura stramonium genetically differentiated in tolerance and resistance to herbivory were used. This set of paternal half-sib families was planted at both sites of origin and the pattern of genotypic selection acting on tolerance and resistance was determined, as well as the presence and variation in the magnitude of allocational costs of tolerance. Selection analyses support the adaptive differentiation hypothesis. Tolerance was favored at the site with higher average level of tolerance, and resistance was favored at the site with higher average level of resistance. The presence of significant environmentally dependent costs of tolerance was in agreement with site variation in the adaptive value of tolerance. Our results support the expectation that environmentally dependent costs of plant defensive strategies can generate differences among populations in the evolutionary trajectory of defensive traits and promote the existence of a selection mosaic. The pattern of contrasting selection on tolerance suggests that, in some populations of D. stramonium, tolerance may alter the strength of reciprocal coevolution between plant resistance and natural enemies.