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Background: Upland Cotton (Gossypium hirsutum L.) has few cotton varieties suitable for mechanical harvesting. The plant height of the cultivar is one of the key features that need to modify. Hence, this study was planned to locate the QTL for plant height in a 60Co γ treated upland cotton semi-dwarf mutant Ari1327. Results: Interestingly, bulk segregant analysis (BSA) and genotyping by sequencing (GBS) methods exhibited that candidate QTL was co-located in the region of 5.80–9.66 Mb at D01 chromosome in two F2 populations. Using three InDel markers to genotype a population of 1241 individuals confirmed that the offspring's phenotype is consistent with the genotype. Comparative analysis of RNA-seq between the mutant and wild variety exhibited that Gh_D01G0592 was identified as the source of dwarfness from 200 genes. In addition, it was also revealed that the appropriate use of partial separation markers in QTL mapping can escalate linkage information. Conclusions: Overwhelmingly, the results will provide the basis to reveal the function of candidate genes and the utilization of excellent dwarf genetic resources in the future. [ABSTRACT FROM AUTHOR]