The prostrate growth habit is an important ornamental trait in ground-cover chrysanthemum, offering high aesthetic value, strong lodging resistance, and excellent landscape greening capability. However, the genetic basis underlying this trait in chrysanthemum remains largely unclear. In this study, we utilized the prostrate-type Chrysanthemum yantaiense (tetraploid), the erect-type C. indicum (tetraploid), and their 199 F₁ hybrid progenies to construct a high-density genetic linkage map through genotyping-by-sequencing. The biparental linkage maps included 4614 and 5180 SNP markers, with an average marker distance of 0.84 and 0.73 cM, respectively. After four years of phenotypic evaluation and one year of dynamic trait measurement in progenies for traits related to prostrate growth habit, we confirmed a stable quantitative trait locus (QTL) located on LG1–1 among co-localized QTLs using KASP markers. This QTL explained up to 20.13% of the phenotypic variation. As a result, a total of 44 genes were identified as candidate due to their tightly linkage with the peak QTL marker, Tag16173. Further phytohormone measurement, gene expression analysis, and transgenic studies confirmed that one of these candidates, the D type cyclin-encoding gene CyCYCD3;1, played a key role in the formation of prostrate growth habit in C. yantaiense. Our results not only enhance the understanding of the molecular mechanisms behind prostrate growth habit but also provide valuable molecular markers for improving plant architecture-related traits in chrysanthemum breeding.

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