Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82–95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.