Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genesUgt5/Ugt5bandβglu-1may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm.This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number ofβglu-1-like,Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40P. glaucaindividuals from eight and nine provenances across Europe and North America respectively.We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity ofβglu-1andUgt5genes. We observed very large copy numbers ofβglu-1, with up to 381 copies in a singleP. glaucaindividual. We observed among-provenance CNV ofβglu-1inP. glaucabut notP. abies.Ugt5bwas predominantly single-copy in both species.This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences.