Map-based gene cloning is a vital strategy for the identification of the quantitative trait loci or genes underlying important agronomic traits. The conventional map-based cloning method is powerful but generally time-consuming and labor-intensive. In this context, we introduce an improved bulked segregant analysis method in combination with a virus-induced gene silencing (VIGS) strategy for rapid and reliable gene mapping, identification and functional verification. This method was applied to a multiple recessive marker line of upland cotton, Texas 582 (T582), and identified unique genomic positions harboring mutant loci, showing the reliability and efficacy of this method. The v1 locus was further fine-mapped. Only one gene, GhCHLI, which encodes one of the subunits of Mg chelatase, was differentially down-regulated in T582 compared with TM-1. A point mutation occurred in the AAA+ conserved region of GhCHLI and led to an amino acid substitution. Suppression of its expression by VIGS in TM-1 resulted in a yellow blade phenotype that was similar to T582. This integrated approach provides a paradigm for the rapid mapping and identification of the candidate genes underlying the genetic traits in plants with large and complex genomes in the future.