Tillers are specialized lateral shoots arising from axillary buds at basal nodes, and are also an important agronomic trait that determines the aboveground biomass and grain yield of various gramineous crops. So far, few genes have been reported to control tiller formation and most have been in the annual crop rice (Oryza sativa). Orchardgrass (Dactylis glomerata) is an important perennial forage crop with great economic and ecological value, but its genes regulating tillering have remained largely unknown. In the present study, we used a natural population of 264 global orchardgrass germplasms to determine genes associated with quantitative variation in tiller number through genome-wide association study analysis. A total of 19 putative loci and 55 genes associated with tiller number were thus identified. Additionally, 26 putative differentially expressed genes with tiller number, including DgCYC-C1, were identified by RNA-seq and genome-wide association study analysis. DgCYC-C1 which is involved in cell division, was overexpressed, revealing that DgCYC-C1 positively regulates tiller number. These results provide some new candidate genes or loci for the improvement of tiller number in crops, which might advance new sustainable strategies to meet global crop production challenges.