Patterns of clinal genetic variation in Drosophila are often characterized after rearing at constant temperatures. However, clinal patterns might change after acclimation if populations differ in their plastic response to fluctuating environments. We studied longevity, starvation and heat knock-down resistance after development at either constant or fluctuating temperatures in nine Drosophila buzzatii populations collected along an altitudinal gradient in Tenerife, Spain. Flies that developed at fluctuating temperatures had higher stress resistance despite experiencing a slightly lower average temperature than those at constant temperatures. Genetic variation along the gradient was found in both stress-resistance traits. Because Q(ST) values greatly exceeded F(ST) values, genetic drift could not explain this diversification. In general, differences among populations were larger after rearing at fluctuating temperatures, especially in heat knock-down, for which clinal patterns disappeared when flies were reared at constant temperatures. This result emphasizes the importance of determining whether populations originating from different environments differ in their plastic responses to stress.