Using bulked segregant analysis based on next-generation sequencing, the recessive nulliplex-branch gene was mapped between two SNP markers ~600 kb apart. In a "nulliplex-branch" cotton mutant, most of the flowers arise directly from leaf axils on the main shoot, which usually does not have a fruiting branch. A nulliplex-branch is a useful trait by which to study cotton architecture; however, the genetic basis of this mutant has remained elusive. In this study, bulked segregant analysis combined with next-generation sequencing technology was used to finely map the underlying genes that result in a nulliplex-branch plant. The nulliplex-branch Pima cotton variety, Xinhai-18, was crossed with the normal branch upland cotton line, TM-1, resulting in an F2 population. The nulliplex-branch trait was found to be controlled by the recessive gene gb_nb1. Allelic single-nucleotide polymorphisms (SNPs) were discovered by reduced-representation sequencing between the parents, and their profiles were also characterized in the nulliplex-branch and normal branch bulks constructed using the F2 plants. A candidate ~9.0 Mb-long region comprising 42 SNP markers was found to be associated with gb_nb1, which helped localize it at the ~600-kb interval on Chr 16 by segregation analysis in the F2 population. The closely linked markers with gb_nb1 developed in this study will facilitate the marker-assisted selection of the nulliplex-branch trait, and the fine map constructed will accelerate map-based cloning of gb_nb1.