Lateral branching is one of the most important traits, which directly determines plant 27 architecture and crop productivity. Commercial watermelon has the characteristics of multiple 28 lateral branches, and it is time-consuming and labor costing to manually remove the lateral 29 branches in traditional watermelon cultivation. In our present study, a lateral branchless trait was 30 identified in watermelon material W CZ, and genetic analysis revealed that it was controlled by a 31 single recessive gene, which named as Clbl . A bulked segregant sequencing (BSA seq) and 32 linkage analysis was conducted to primarily mapping of Clbl on watermelon chromosome 4 33 Next-generation sequencing aided marker discovery and a large mapping population consisting of 34 1406 F 2 plants was used to further mapped the Clbl locus into a 9011 bp candidate region which 35 harbored only one candidate gene Cla018392 encoding a TERMINAL FLOWER 1 gene. Sequence 36 comparison of Cla018392 between two parental lines revealed that there was a SNP detected from 37 C to A in the coding region in the branchless inbred line WCZ , which resulted in a mutation of 38 Alanine (GCA) to Glutamate (GAA) at the fourth exon A dCAPS marker was developed from the 39 SNP locus, which was co-segregated with the branchless phenotype in both BC 1 and F 2 population, 40 and it was also further validated in 152 natural watermelon accessions. qRT PCR and in situ 41 hybridization showed that the expression levels of Cla0 18392 was significantly reduced in the 42 axillary bud and apical bud in the branchless line WCZ Ectopic expression of ClTFL1 in 43 Arabidopsis showed an increased number of lateral branches. The results of this study will be 44 useful for better understanding the molecular mechanism of lateral branch development in 45 watermelon and for the development of marker-assisted selection (MAS) for new branchless 46 watermelon cultivars.