Forest trees face threats from many insect pest species, underscoring the importance of understanding their defence mechanisms for survival. In a North American conifer species, Picea glauca, gene expression of βglu-1 produces phenolic compounds (acetophenones) that defend against its insect defoliator, Choristoneura fumiferana. βglu-1 is also expressed in a Eurasian conifer species, Picea abies, although no major insect defoliators are present within the species' natural range. We compared the range-wide variation of βglu-1 transcript levels from foliage samples of P. glauca in North America and P. abies in Europe using RT-qPCR and targeted transcriptome sequencing. βglu-1 transcript levels were highly correlated between the two methods, with large transcript level variation being detected within and between populations in both species and one of the βglu-1 gene forms being significantly differentially expressed in P. glauca. Importantly, βglu-1 transcript levels in P. glauca varied longitudinally and were positively associated with mean annual precipitation and C. fumiferana outbreak class, which has historically higher outbreak frequency and severity in eastern compared to western populations. In P. abies, βglu-1 transcript levels were associated with annual temperature range. Overall, these results enhance our understanding of potential adaptive variation in acetophenone defences in P. glauca, while the factors influencing βglu-1 transcript variation in P. abies require further investigation.