Forest trees face threats from many insect pest species, underscoring the importance of understanding their defense mechanisms for survival. In a North American conifer species Picea glauca, white spruce, a defense-related gene, βglu1, is responsible for releasing phenolic compounds (acetophenones) to defend against its insect defoliator, Choristoneura fumiferana, the eastern spruce budworm. βglu1 is also expressed in a Eurasian conifer species Picea abies, Norway spruce, although no major insect defoliator is present within the species’ natural range. We compared range-wide variation of βglu1 transcript levels from foliage samples of P. glauca in North America and P. abies in Europe using RT-qPCR and targeted transcriptome sequencing. βglu1 transcript levels were highly correlated between the two methods, with wide ranges of variation being detected within and between populations in both species. We found a significant longitudinal gradient in βglu1 transcript levels in P. glauca, with one βglu1 gene form being differentially expressed across populations, but not in P. abies. The expression level differences in P. glauca are consistent with the historically higher C. fumiferana outbreak frequency and severity in eastern compared to western populations, with C. fumiferana defoliation severity being a significant explanatory variable for βglu1 transcript levels. Climate per se was not a significant explanatory factor in either species. Overall, these results enhance our understanding of potential adaptive variation in acetophenone defenses in P. glauca, while the factors influencing βglu1 transcript variation in P. abies require further investigation.