Plant geneticists have proposed that the dynamic conservation of crop plants in farm environments (in situ conservation) is complementary to static conservation in seed banks (ex situ conservation) because it may help to ensure adaptation to changing conditions. Here, we test whether collections of a traditional variety of Moroccan barley (Hordeum vulgare ssp. vulgare) conserved ex situ showed differences in qualitative and quantitative resistance to the endemic fungal pathogen, Blumeria graminis f.sp. hordei, compared to collections that were continuously cultivated in situ. In detached-leaf assays for qualitative resistance, there were some significant differences between in situ and ex situ conserved collections from the same localities. Some ex situ conserved collections showed lower resistance levels, while others showed higher resistance levels than their in situ conserved counterparts. In field trials for quantitative resistance, similar results were observed, with the highest resistance observed in situ. Overall, this study identifies some cases where the Red Queen appears to drive the evolution of increased resistance in situ. However, in situ conservation does not always result in improved adaptation to pathogen virulence, suggesting a more complex evolutionary scenario, consistent with several published examples of plant–pathogen co-evolution in wild systems.