As in most insect groups, host plant shifts in cactophilic Drosophila represent environmental challenges as flies must adjust their developmental programme to the presence of different chemical compounds and/or to a microflora that may differ in the diversity and abundance of yeasts and bacteria. In this context, wing morphology provides an excellent opportunity to investigate the factors that may induce changes during development. In this work, we investigated phenotypic plasticity and developmental instability of wing morphology in flies on the cactophilic Drosophila buzzatii and Drosophila koepferae raised on alternative breeding substrates. We detected significant differences in wing size between and within species, and between flies reared on different cactus hosts. However, differences in wing shape between flies emerged from different cactus hosts were not significant either in D. buzzatii or in D. koepferae. Our results also showed that morphological responses involved the entire organ, as variation in size and shape correlated between different portions of the wing. Finally, we studied the effect of the rearing cactus host on developmental instability as measured by the degree of fluctuating asymmetry (FA). Levels of FA in wing size were significantly greater in flies of both species reared in non-preferred when compared with those reared in preferred host cacti. Our results are discussed in the framework of an integrative view aimed at investigating the relevance of host plant shifts in the evolution of the guild of cactophilic Drosophila species that diversified in South America.