Lignin metabolism plays a pivotal role in plant defense against pathogens and is always positively correlated as a response to pathogen infection.  Thus, understanding resistance genes against pathogens in plants depends on a genetic analysis of lignin response.  In the study, eight upland cotton lines were used to construct a multi-parent advanced generation intercross (MAGIC) population (n=280), which exhibited peculiar characteristics from the convergence of various alleles coding for advantageous traits.  To measure the lignin response to Verticillium wilt (LRVW), artificial disease nursery (ADN) and rotation nursery (RN) were prepared for MAGIC population planting in four environments.  The stem lignin contents were collected, and the LRVW was measured with the lignin value of ADN/RN in each environment, which showed great variation.  A total of 9323 high-quality single-nucleotide polymorphism (SNP) markers obtained from the Cotton-SNP63K array were employed for genotyping the MAGIC population.  The SNPs were distributed through the whole genome with 4.78 SNP/Mb density, ranging from 1.14 (ChrA06) to 10.08 (ChrD08).  A genome-wide association study was performed using a mixed linear model (MLM) for LRVW, and three stable quantitative trait loci (QTLs), qLRVW-A04, qLRVW-A10 and qLRVW-D05, were identified in more than two environments.  Two key candidate genes, Ghi_D05G01046 and Ghi_D05G01221, were selected within the QTLs through the combination of variations in the coding sequence, induced expression patterns, and function annotations, both of which presented nonsynonymous mutations in coding regions and were strongly induced by Verticillium dahliae. Ghi_D05G01046 encodes a leucine-rich extensin (LRx) protein, which is involved in Arabidopsis cell wall biosynthesis and organization.  Ghi_D05G01221 encodes a transcriptional co-repressor novel interactor of jaz (NINJA), which functions in the jasmonic acid (JA) signaling pathway.  In summary, the study creates valuable genetic resources for breeding and QTL mapping and opens up a new perspective to uncover the genetic basis of VW resistance in upland cotton.