Branching number (BN) is a crucial architectural trait in Cucumis melo. Because of its multiple branch habits, much more labour costs are needed in melon production. However, the genetic mechanism of branching numbers in melon is not clear. Here, a genetic population from multiple branching material S8 (only two branching number in the first node) as the female line and S7 (multiple branching numbers in each node; more than nine branch numbers) as the male parent is used to make a cross F2:3 generation. By performing QTL mapping based on bulked segregate analysis (BSA) after two years, a candidate QTL region of the BN was located on chromosome 3. For further QTL mapping, a genetic linkage map, which contained 16 SSR markers with a total length of 2.27 Mb, was constructed. One major QTL locus bnDQ-2022-3.1 was detected between CmSSR9556 and CmSSR9580, with a LOD threshold of 11.37 and a contribution rate of 49.11% in the spring of 2022 in Daqing City. Then, a consistent QTL bnSY-2022-3.1 was also investigated in Sanya, Hainan Province, in the autumn of 2022, with a LOD threshold of 10.85 and a contribution rate of 45.01%. Nine genes were investigated within the interval of the candidate region located in chromosome 3 between 22,723,436 and 22,807,889 of the melon’s physical position within the 85.45 kb length region. Gene expression analysis showed significant differences between MELO3C019872.2.1, MELO3C030060.2.1, and MELO3C019871.2.1 in different development stages. Gene sequence different analysis revealed a “C”-to-“T” mutation in the 1280 bp site of MELO3C030060.2.1 in parental lines. Heterologous transformation of MELO3C030060.2.1 into cucumber revealed that overexpression of MELO3C030060.2.1 resulted in more and denser branches in cucumber plants, and the growth rate of lateral branches was significantly faster than that of the wild type. Transferring to antisense of MELO3C030060.2.1 had the opposite effect. To sum up, MELO3C030060.2.1 is related to melon branching initial habits. This study could provide a new insight into melon branching habits and provide a theoretical base for melon breeding.
