A characteristic feature of plant cells is the ability to form callus from parenchyma cells in response to biotic and abiotic stimuli. Tissue culture propagation of recalcitrant plant species and genetic engineering for desired phenotypes typically depends on efficient in vitro callus generation. Callus formation is under genetic regulation, and consequently, a molecular understanding of this process underlies successful generation for propagation materials and/or introduction of genetic elements in experimental or industrial applications. Herein, we identified 11 genetic loci significantly associated with callus formation in Populus trichocarpa using a genome-wide association study (GWAS) approach. Eight of the 11 significant gene associations were consistent across biological replications, exceeding a chromosome-wide–log10 (p) = 4.46 [p = 3.47E−05] Bonferroni-adjusted significance threshold. These eight genes were used as hub genes in a high-resolution co-expression network analysis to gain insight into the genome-wide basis of callus formation. A network of positively and negatively co-expressed genes, including several transcription factors, was identified. As proof-of-principle, a transient protoplast assay confirmed the negative regulation of a Chloroplast Nucleoid DNA-binding-related gene (Potri.018G014800) by the LEC2 transcription factor. Many of the candidate genes and co-expressed genes were 1) linked to cell division and cell cycling in plants and 2) showed homology to tumor and cancer-related genes in humans. The GWAS approach based on a high-resolution marker set, and the ability to manipulate targets genes in vitro, provided a catalog of high-confidence genes linked to callus formation that can serve as an important resource for successful manipulation of model and non-model plant species, and likewise, suggests a robust method of discovering common homologous functions across organisms.