Coniferous species are mostly monoecious. Their usage in forest plantations is largely limited by the lowunstable yields of seed cones. Gibberellins (GAs)ethylene are central gender regulators in flowering, although knowledge about their metabolism is limited in conifers. Here, we explore gender determination regulators, especially GAethylene metabolism genes, in Qinghai spruce (Picea crassifolia Kom.) by transcriptome analysis of female, malevegetative buds during bud formationdevelopment. Year-round observations were performed to determine the window of bud differentiation. Samples of buds beforeafter differentiation were collected for RNA sequencing, which showed that the bud gender determination process occurred before the morphological differentiation of female, malevegetative buds. Sixteen homologs of GA metabolism genes were identified in the transcriptome. Concentrations of GA1, GA3, GA9,GA15 were identifiedquantified using ultra-high-performance liquid chromatography—high-resolution mass spectrometry (UPLC-HRMS). Furthermore, phylogenetic analysis was performed on 9 putative ACC synthesis (ACS) genes identified in the transcriptomes,quantitative real-time PCR was conducted to examine gene expression. Our study provides information on GAethylene metabolism genes in female, malevegetative buds during bud differentiation. Overall, fine-tuned regulation of GA metabolism contributes to both the reproductive transitiongender differentiation in this species, whereas ethylene might affect male cone bud formation. The current findings could further our knowledge about the regulatory mechanisms underlying the gender differentiation of cone buds.